Annealing control primer and its uses

ABSTRACT

The present invention relates to an annealing control primer for improving annealing specificity in nucleic acid amplification and its applications to all fields of nucleic acid amplification-involved technology. The present primer comprises (a) a 3′-end portion having a hybridizing nucleotide sequence substantially complementary to a site on a template nucleic acid to hybridize therewith; (b) a 5′-end portion having a pre-selected arbitrary nucleotide sequence; and (c) a regulator portion positioned between said 3′-end portion and said 5′-end portion comprising at least one universal base or non-discriminatory base analog, whereby said regulator portion is capable of regulating an annealing portion of said primer in association with annealing temperature.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/402,980, filed Feb. 23, 2012, which is a continuation of U.S.application Ser. No. 12/458,702, filed Jul. 21, 2009, which is acontinuation of U.S. application Ser. No. 11/651,605, filed Jan. 10,2007, now U.S. Pat. No. 7,579,154, which is a continuation-in-part ofU.S. application Ser. No. 10/269,031, filed Oct. 11, 2002, which is acontinuation-in-part of U.S. application Ser. No. 10/014,496, filed Dec.14, 2001 and claims priority to PCT application No. PCT/KR01/02133,filed Dec. 8, 2001, all of which are herein incorporated by reference intheir entirety.

DESCRIPTION OF THE FILES CONTAINED ON THE CD-R

The contents of the submission on compact discs submitted inprior-related application Ser. No. 11/651,605, filed Jan. 10, 2007 (nowU.S. Pat. No. 7,579,154, issued Aug. 25, 2009) are incorporated hereinby reference in their entirety: A compact disc copy of the SequenceListing (COPY 1) (filename: SEEG 001 02US SeqList.txt, date recorded:Jan. 10, 2007, file size 33 kilobytes); a duplicate compact disc copy ofthe Sequence Listing (COPY 2) (filename: SEEG 001 02US SeqList.txt, daterecorded: Jan. 10, 2007, file size 33 kilobytes); a computer readableformat copy of the Sequence Listing (CRF COPY) (filename: SEEG 001 02USSeqList.txt, date recorded: Jan. 10, 2007, file size 33 kilobytes). Thepaper copy of the Sequence Listing is filed herewith and incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to an annealing control primer and itsapplications. More particularly, the present invention relates to anannealing control primer for improving annealing specificity in nucleicacid amplification and its applications to all fields of nucleic acidamplification-involved technology.

DESCRIPTION OF THE RELATED ART

Nucleic acid amplification is a pivotal process for a wide variety ofmethods in molecular biology, so that various amplification methods havebeen proposed. For example, Miller, H. I. et al. (WO 89/06700) disclosea nucleic acid sequence amplification based on the hybridization of apromoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Otherknown nucleic acid amplification procedures include transcription-basedamplification systems (Kwoh, D. et al., Proc. Natl. Acad. Sci. U.S.A.,86:1173(1989); and Gingeras T. R. et al., WO 88/10315).

Schemes based on ligation of two or more oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, arealso known (Wu, D. Y. et al., Genomics 4:560 (1989)), which are called“Ligation Chain Reaction” (LCR).

Davey, C. et al. (European Pat. Appln. Publication No. 329,822) disclosea nucleic acid amplification process involving cyclically synthesizingsingle-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA).The ssRNA is a first template for a first primer oligonucleotide, whichis elongated by reverse transcriptase (RNA-dependent DNA polymerase).The RNA is then removed from resulting DNA:RNA duplex by the action ofribonuclease H. The resultant ssDNA is a second template for a secondprimer, which also includes the sequences of an RNA polymerase promoter.This primer is then extended by DNA polymerase, resulting as adouble-stranded DNA (“dsDNA”) molecule, having a sequence identical tothat of the original RNA between the primers and having additionally, atone end, a promoter sequence. This promoter sequence can be used by theappropriate RNA polymerase to produce many RNA copies of the DNA. Thesecopies can then re-enter the cycle leading to very rapid amplification.

The most predominant process for nucleic acid amplification known aspolymerase chain reaction (hereinafter referred to as “PCR”), is basedon repeated cycles of denaturation of double-stranded DNA, followed byoligonucleotide primer annealing to the DNA template, and primerextension by a DNA polymerase (Mullis et al. U.S. Pat. Nos. 4,683,195,4,683,202, and 4,800,159; Saiki et al. 1985). The oligonucleotideprimers used in PCR are designed to anneal to opposite strands of theDNA, and are positioned so that the DNA polymerase catalyzed extensionproduct of one primer can serve as the template strand for the otherprimer. The PCR amplification process results in the exponentialincrease of discrete DNA fragments whose length is defined by the 5′ends of the oligonucleotide primers.

The success in the nucleic acid amplifications, in particular PCRamplification, relies on the specificity with which a primer annealsonly to its target (and not non-target) sequences and therefore it isimportant to optimize this molecular interaction. Whether a primer cananneal only to its perfect complement or also to sequences that have oneor more mismatches, depends critically upon the annealing temperature.In general, the higher the annealing temperature, the more specificannealing of the primer to its perfect matched template and so thegreater the likelihood of only target sequence amplification can beaccomplished. The lower the temperature, the more mismatches betweentemplate and primer can be tolerated, leading to increased amplificationof non-target sequences. Adjusting the annealing temperature can alterthe specificity of pairing between template and primer. For examples, ifthere is no product, the temperature may be too high and can be reduced.If there are products in control where only one primer is present, thisindicates that the single primer is annealing to more than one region ofthe template. In this case, the annealing temperature should beincreased. Considering such effect of annealing temperature on primerannealing specificity, there remains a strong need for an annealingcontrol primer system which is capable of controlling primer annealingin accordance with annealing temperature to enhance primer annealingspecificity regardless of primer design.

In addition to annealing temperature, several “primer search parameters”such as primer length, GC content and PCR product length (Dieffenbach etal., 1995) should be considered for primer annealing specificity. If aprimer, which satisfies all such parameters, were employed, primerannealing would be specified, resulting in the significant enhancementof primer annealing specificity during target DNA amplification and thefreedom from the problems such as backgrounds and non-specific productsarising from primers used in the experiments. It is usual thatwell-designed primers can help avoid non-specific annealing andbackgrounds as well as distinguish between cDNAs or genomic templates inRNA-PCR.

Many approaches have been developed to improve primer annealingspecificity and therefore accomplish the amplification of the desiredproduct. Examples are touchdown PCR (Don et al., 1991), hot start PCR(D'Aquila et al., 1991), nested PCR (Mullis and Faloona, 1987) andbooster PCR (Ruano et al., 1989). Another alternative approaches havebeen also reported that various ‘enhancer’ compounds can improve thespecificity of PCR. The enhancer compounds include chemicals thatincrease the effective annealing temperature of the reaction, DNAbinding proteins and commercially available reagents. However, there isno ‘magic’ additive that will ensure the success in every PCR and it isvery tedious to test different additives under different conditions suchas annealing temperature. Although these approaches have contributed tothe improvement of primer annealing specificity in some cases, they havenot accessed fundamentally to a solution for the problems arising fromprimers used in the PCR amplification, such as non-specific products andhigh backgrounds.

In many cases, the primer sequence does not need to be a perfectcomplement to the template sequence. The region of the primer thatshould be perfectly matched to the template is the 3′-end because thisend is the region of the primer extended by the DNA polymerase and istherefore the most important for ensuring the specificity of annealingto the correct target sequence. The 5′-end of the primer is lessimportant in determining specificity of annealing to the target sequenceand can be modified to carry additional sequence such as restrictionsites and promoter sequences that are not complementary to the template(McPherson and Moller 2000). This notion is adapted to the design of theannealing control primers of this invention as described below.

PCR-based techniques have been widely used not only for amplification ofa target DNA sequence but also for scientific applications or methods inthe fields of biological and medical research such as Reversetranscriptase PCR (RT-PCR), Differential Display PCR (DD-PCR), Cloningof known or unknown genes by PCR, Rapid amplification of cDNA ends(RACE) and PCR-based genomic analysis (McPherson and Moller, 2000). Thefollowings are only representatives of PCR applications.

Techniques designed to identify genes that are differentially regulatedby cells under various physiological or experimental conditions (forexample, differentiation, carcinogenesis, pharmacological treatment)have become pivotal in modern biology. One such method for screeningdifferences in gene expression between various cell types or betweendifferent stages of cell development with the availability of PCR isknown as Differential Display PCR (DD-PCR), described by Liang andPardee in 1992. This method uses combinations of 10-mer arbitraryprimers with anchored cDNA primers and generates fragments thatoriginate mostly from the poly(A) tail and extend about 50-600nucleotide upstream. By combining 3′ anchored Oligo(dT) primers andshort 5′ arbitrary primers, the subsets of the transcriptome areamplified, the resulting cDNA fragments are generally separated ondenaturing polyacrylamide gel and visualized autoradiographically.

Although this method is simple and rapid and only requires small amountsof total RNA, there are a number of disadvantages in the conventionalDD-PCR methods. The differential banding patterns are often only poorlyreproducible due to the use of short arbitrary primer so that manylaboratories have had difficulty in obtaining reproducible results withthese methods. It has been shown that at least 40% of the differentiallydisplayed bands are not reproducible between experiments even inwell-trained hands (Bauer et al., 1994). Furthermore, the pattern ofdifferential expression often cannot be reproducible on Northern blotsand the percentage of these false positives can arise up to 90%(Sompayrac et al., 1995). As a modification used for an alternative, theuse of longer random primers of, e.g. 20 bases in length does notsatisfactorily solve the problem of reproducibility (Ito et al., 1994).There are another factors responsible for the relatively lowreproducibility of DD-PCR such as an insufficient amount of startingmaterial and very low concentration of dNTP (2-5 μM) employed to preparethe different banding patterns (Matz and Lukyanov, 1998). It is alsodifficult to detect rare transcripts with these methods (Matz andLukyanov, 1998). In addition, because the cDNA fragments obtained fromDD-PCR are short (typically 100-500 bp) and correspond to the 3′-end ofthe gene that represent mainly the 3′ untranslated region, they usuallydo not contain a large portion of the coding region. Therefore, thelabor-intensive full-length cDNA screening is needed unless significantsequence homology, information for gene classification and prediction offunction is obtained (Matz and Lukyanov, 1998).

Differential Display methods generally use radioactive detectiontechniques using denaturing polyacrylamide gels. The radioactivedetection of the reaction products restricts the use of this techniqueto laboratories with the appropriate equipment. Relatively long exposuretimes and problems with the isolation of interesting bands from thepolyacrylamide gels are additional drawbacks of Differential Displaytechnique. Although modified non-radioactive Differential Displaymethods have recently been described, which include silver staining(Gottschlich et al. 1997; Kociok et al., 1998), fluorescent-labeledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), the use of biotinylated primers (Korn et al.,1992; Tagle et al., 1993; Rosok et al., 1996) and ethidiumbromide-stained agarose gels (Rompf and Kahl, 1997; Jefferies et al.,1998; Gromova et al., 1999), these methods have met with only limitedsuccess. If the reaction products could be simply detected on ethidiumbromide-stained agarose gel and the results were reproducible andreliable, it would greatly increase the speed of DD-PCR analysis andavoid the use of radioactivity.

Another PCR-based approach called targeted differential display uses anoligonucleotide primer that directs the amplification of multigenefamily members with conserved protein domains. Gene families are groupsof genes which are often functionally characterized by a particular typeof function undertaken by the gene products in a cell and whichstructurally have one or more conserved regions (domains) in common.Examples of gene families include the MADS-box and the homeogene familyas well as further transcription factor families. The cyclin, cytokineand globin gene families are examples of medical interest. The PrositeDatabase provides a list of proteins that have common domains andsequence motifs. The oligonucleotide used in the PCR can either be aspecific primer that is used at a low annealing temperature or, as ismore often the case, a degenerate primer mixture for use at higherstringencies (Stone and Wharton, 1994). However, amplifications usingdegenerate primers can sometimes be problematic and may requireoptimization. It is important to keep the annealing temperature as highas possible to avoid extensive nonspecific amplification and a good ruleof thumb is to use 55° C. as a starting temperature. In general, it isdifficult to keep this rule because degenerate primers should bedesigned on the basis of amino acid sequences or conserved domainsequences as a precondition. In order to generate a satisfiedrelationship between degenerate primer and annealing temperature in thisapproach, it is required to use an annealing control primer which cantolerate the alternation of annealing temperature, particularly hightemperature such as 68° C. regardless of primer design.

Still another PCR-based technique is arbitrary primed PCR (AP-PCR) forRNA fingerprinting. One great strength of AP-PCR methods is theirsimplicity (Welsh and McClelland, 1991; Williams et al., 1990). AP-PCRuses a single primer or a pair of primers, wherein the primers are10-mers or 18-mers as longer primer. This method has previously beenused to provide DNA fingerprints of hybrid cell lines (Ledbetter et al.,1990) and particular genomic regions (Welsh and McClelland, 1990;Williams et al., 1990). It provides a very useful tool for genomeanalysis in bacteria, fungi and plant identification and populationstudies, where individual isolates can be compared rapidly. For example,they can be used as a tool to identify pathogens or the occurrence ofparticular strains or pathotypes. Commonly, AP-PCR uses a single primerto initiate DNA synthesis from regions of a template where the primermatches imperfectly. In order for this to work, the initial cycles haveto be performed at low stringency (37-50° C.), normally for the firstfive cycles, which allows primer annealing to imperfect sites throughoutthe genome. The stringency is then increased (55° C.) as for standardPCR amplification and the reaction is allowed for an additional 30-35cycles. AP-PCR is not recommended for use in such applications aspaternity testing where unequivocal results are demanded, becausenonparental products are occasionally produced. Although alternativeAP-PCR approaches including nested AP-PCR have been developed(McClelland et al., 1993; Ralph et al., 1993), the issue ofreproducibility is still of main concern. One concern is that thepatterns may vary from day to day or from lab to lab (see, e.g., Meunierand Grimont, 1993).

Still yet another PCR-based application is RACE (rapid amplification ofcDNA end) technology. RACE is a procedure for amplification of cDNAregions corresponding to the 5′- or 3′-end of mRNA (Frohman et al.,1988) and it has been used to isolate rare transcripts successfully. Thegene-specific primer may be derived from sequence data from a partialcDNA, genomic exon or peptide. In 3′ RACE, the polyA tail of mRNAmolecules is exploited as a priming site for PCR amplification. mRNAsare converted into cDNAs using reverse transcriptase and an Oligo-dTprimer as known in the art. The generated cDNAs can then be directly PCRamplified using a gene-specific primer and a primer that anneals to thepolyA region.

The same principle as 3′ RACE applies to 5′ RACE but there is no polyAtail. Thus, 5′ RACE is made by tagging the 5′-end of a cDNA by means ofdifferent methods (Fromont-Racine et al., 1993; Schaefer, 1995; Franz etal., 1999). Most approaches for the 5′ RACE such as homopolymerictailing and ligation anchored tailing require a set of enzymaticreactions after completion of first strand cDNA synthesis (Schaefer,1995). Each enzymatic step has the potential to introduce failures andto destroy the integrity of the cDNA. Recently, an alternative has beenintroduced, the so-called CapFinder approach (Chenchik et al., 1998;Chenchik et al. U.S. Pat. Nos. 5,962,271 and 5,962,272). The techniquerelies on dual functions of the reverse transcriptases: one is theterminal transferase activity to add non-templated nucleotides to the3′-end of a cDNA and the other is the template switching activity toswitch a template to a second template. This property is utilized duringthe retroviral life cycle (Clark, 1988; Kulpa et al., 1997). Moloneymurine leukemia virus (M-MLV) reverse transcriptase (RT) often addsthree to four non-template-derived cytosine residues to the 3′-end ofnewly synthesized cDNAs in the presence of manganese or high magnesium(Schmidt and Mueller, 1999). This approach allows the amplification offull-length cDNAs because the M-MLV RT adds C residues preferentially tothe cDNA if complete (capped) mRNA serves as template.

However, the CapFinder approach for 5′-RACE experiments could not befree from background problems such as DNA smear arising from thecontamination of the CapFinder and Oligo-dT primers, which are used incDNA synthesis (Chenchik et al., 1998). Even residual amounts of theseprimers result in a high background because both ideally fit to allcDNAs present in the reaction mixture. In addition, 3′-RACE andfull-length cDNA amplification have the same background problems due tothe contamination of primers used for cDNA synthesis in which theygenerate non-specific products in PCR reaction (Chenchik et al., 1998).New approaches to overcome the problems above have been recentlyintroduced. One approach is step-out PCR to suppress unwanted PCRproducts (Matz et al., 1999) but it has been pointed out that thisapproach still remains a smear of DNA rather than a single DNA (Schrammet al., 2000). Another approach which is introduced more recently is touse solid-phase cDNA synthesis and procedures to remove all contaminantsused in cDNA synthesis (Schramm et al., 2000), but the major drawback ofthis technique is costly and time-consuming by requiring solid-phasecDNA synthesis and following procedures. Therefore, more effective,simple, rapid and inexpensive strategies are required to completelyeliminate problems arising from contamination of the primers such asOligo-dT or CapFinder primer used for cDNA synthesis.

In addition to RACE technologies, in current technologies for cDNAlibrary construction, the 5′-ends of genes tend to be under-representedin cDNA populations, especially where a poly(dT) primer is used duringfirst cDNA strand synthesis and the starting material is limited.Although a number of different approaches have been developed toovercome this problem, most suffer from common limitations producingfull-length cDNAs or 5′-enriched cDNAs with a number of inherentproblems. These approaches are complex or costly and time-consuming byrequiring multiple enzymatic steps and/or are not pronounced sensitive(Carninci et al., 1997; Suzuki et al., 1997; Guegler et al. U.S. Pat.Nos. 6,083,727 and 6,326,175; Hayashizaki. U.S. Pat. No. 6,143,528).Therefore, there is continued interest in the development of improvedmethods for generating full-length or 5-enriched cDNAs, particularlywith the limited starting material.

Multiplex PCR is another variant of PCR in which more than one targetsequence can be simultaneously amplified with more than one pair ofprimers in the same reaction. Since its first description in 1988(Chamberlain et al., 1988), this method has been successfully applied inmany areas of DNA testing, including analyses of gene deletion(Anonymous, 1992; Henegariu, et al., 1994), mutation and polymorphismanalysis (Shuber et al., 1993; Mutirangura et al., 1993), quantitativeanalysis (Zimmermann et al., 1996), and RNA detection (Zou et al.,1998). In the field of infection diseases, the technique has been shownto be a valuable method for identification of viruses, bacteria, fungi,and/or parasites.

However, the results obtained with multiplex PCR are frequentlycomplicated by the artifacts of the amplification procedure. Theseinclude “false-negative” results due to reaction failure and“false-positive” results such as the amplification of spurious products,which may be caused by annealing of the primers to sequences which arerelated to but distinct from the true recognition sequences. For use inmultiplex PCR, a primer should be designed so that its predictedhybridization kinetics are similar to those of the other primers used inthe sample multiplex reaction. While the annealing temperature andprimer concentrations may be calculated to some degree, the conditionsgenerally have to be empirically determined for each multiplex reaction.Since the possibility of non-specific priming increases with eachadditional primer pair, the conditions must be modified as necessary asindividual primer sets are added. Moreover, the artifacts that resultfrom competition for resources (e.g., depletion of primers) areaugmented in multiplex PCR, since the differences in the yields ofunequally amplified fragments are enhanced with each cycle. Thus, theoptimization of the reaction conditions for multiplex PCR can becomelabor-intensive and time-consuming. Since the different multiplex PCRsmay have unique reaction conditions, the development of new diagnostictests can become very costly.

Therefore, there is a need in the art for primers that allow multiplexPCR reactions to be designed and carried out without elaborateoptimization steps, irrespective of the potentially divergent propertiesof the different primers used. Furthermore, there is a need in the artfor primers that allow multiplex PCR reactions that, under the samereaction conditions, simultaneously produce equivalent amounts of eachof many amplification products.

Single nucleotide polymorphisms (SNPs), the most common geneticvariations found in the human genome, are important markers foridentifying disease-associated loci and for pharmaco-genetic studies(Landegren et al., 1998; Roses, 2000). SNPs appear in the human genomewith an average of once every 1000 by and totaling >3 million. A varietyof approaches have been used to detect SNPs. However, one of the keybottlenecks is the amplification of DNA. Most current assays include astep that produces many copies of a short segment of the sample DNAspanning each target SNP. This amplification is usually necessarybecause only small amounts of DNA can be harvested from typical clinicalsamples. Also, the amplification improves the signal-to-noise ratio ofthe assays, increasing the reliability of detection. Most genotypingtechniques accomplish this amplification using PCR. Most importantly,the specificity of PCR amplification is critical in the application ofPCR in the SNP genotyping. Therefore, it would be beneficial if themethods for improving PCR specificity are available and applied to thedevelopment of SNP genotyping assay. It would also be beneficial if suchmethods are capable of providing multiple analyses in a single assay(multiplex assays).

As described above, all these methods and techniques involving nucleicacid amplification, in particular PCR amplification, could not becompletely free from the limitations and problems resulting from thenon-specificity of primers used in each method, such as false positives,poor reproducibility, high backgrounds and so on, although improvedapproaches to each method has been continuously introduced. Therefore,there remains a need of novel primer for improving annealing specificityand methods, which can give rise to true results.

Throughout this application, various patents and publications arereferenced and citations are provided in parentheses. The disclosure ofthese patents and publications in their entities are hereby incorporatedby references into this application in order to more fully describe thisinvention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

Endeavoring to resolve the problems of such conventional primer andvarious methods involving nucleic acid amplification, the presentinventor has developed a novel annealing control primer that can permitnucleic acid amplification with much higher specificity and itsunlimited applications in all fields of nucleic acid amplification-basedtechnology.

Accordingly, it is an object of this invention to provide an annealingcontrol primer for improving annealing specificity in nucleic acidamplification.

It is another object of this invention to provide a method foramplifying a nucleic acid sequence from a DNA or a mixture of nucleicacids as template.

It is still another object of this invention to provide a method forselectively amplifying a target nucleic acid sequence from a DNA or amixture of nucleic acids as template

It is further object of this invention to provide a method forselectively amplifying a target nucleic acid sequence from an mRNA.

It is still further object of this invention to provide a method fordetecting DNA complementary to differentially expressed mRNA in two ormore nucleic acid samples.

It is another object of this invention to provide a method for rapidlyamplifying a target cDNA fragment comprising a cDNA region correspondingto the 3′-end region of an mRNA.

It is still another object of this invention to provide a method foramplifying a target cDNA fragment comprising a cDNA region correspondingto the 5′-end region of an mRNA.

It is further object of this invention to provide a method foramplifying a population of full-length double-stranded cDNAscomplementary to mRNAs.

It is still further object of this invention to provide a method foramplifying 5′-enriched double-stranded cDNAs complementary to mRNAs.

It is another object of this invention to provide a method foramplifying more than one target nucleotide sequence simultaneously.

It is still another object of this invention to provide a method forproducing a DNA fingerprint of gDNA.

It is still another object of this invention to provide a method forproducing a RNA fingerprint of an rnRNA sample.

It is further object of this invention to provide a method foridentifying a conserved homology segment in a multigene family.

It is still further object of this invention to provide a method foridentifying a nucleotide variation in a target nucleic acid.

It is another object of this invention to provide a method formutagenesis in a target nucleic acid.

It is still another object of this invention to provide a kit comprisingan annealing control primer.

It is further object of this invention to provide kits for a variety ofmethods involving nucleic acid amplification.

It is still further object of this invention to provide a use of anannealing control primer for a process involving nucleic acidamplification.

Other objects and advantages of the present invention will becomeapparent from the detailed description to follow taken in conjugationwith the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show schematic representations for selectivelyamplifying a target nucleic acid of double-stranded DNA (1A) or mRNA(1B) using the ACP of the present invention.

FIGS. 2A and 2B show schematic representations for identifyingdifferentially expressed genes using the ACP of the present invention.

FIG. 3 shows a schematic representation for amplifying a target cDNAfragment comprising 3′-end region corresponding to the 3′-end of mRNAusing the ACP of the present invention.

FIGS. 4A and 4B show schematic representations for amplifying a targetcDNA fragment comprising 5′-end region corresponding to the 5′-end ofmRNA using the ACP of the present invention. The Oligo dT (4A) or randomprimer (4B) is used as a first-strand cDNA synthesis primer.

FIG. 5 shows a schematic representation for amplifying full-length cDNAmolecules complementary to the mRNA molecules using the ACP of thepresent invention.

FIG. 6 shows a schematic representation for amplifying 5′ enriched cDNAmolecules complementary to the mRNA molecules comprising the 5′-endinformation using the ACP of the present invention.

FIG. 7A shows a schematic representation for detecting single nucleotidepolymorphism (SNP) using the ACP of the present invention.

FIG. 7B shows another schematic representation for detecting singlenucleotide polymorphism (SNP) using the ACP of the present invention.

FIG. 8 is an agarose gel photograph to show the effect of a deoxyinosinegroup positioned between the 3′- and 5′-end portions of ACP. The cDNAwas amplified using total RNA isolated from conceptus tissues at E4.5(lanes 1 and 4), E11.5 (lanes 2 and 5), and E18.5 (lanes 3 and 6), witha set of the dTIO-JYC2 (SEQ ID NO. 29) and ACP10 (lanes 1-3) (SEQ ID NO.13), and a set of the dT10-ACP1 (SEQ ID NO. 30) and ACP10 (lanes 4-6),respectively.

FIG. 9 is an agarose gel photograph to show the effect of deoxyinosineresidues positioned between the 3′- and 5′-end portions of ACP inassociation with the alteration of number of deoxyinosine during PCR.The lanes 0, 2, 4, 6, and 8 represent the number of deoxyinosineresidues, respectively.

FIG. 10A is an agarose gel photograph to show the results of two stagePCR amplifications for Esx1 using a set of EsxN7 and EsxC6 primers(lane 1) and a set of EsxN7-ACP and EsxC6-ACP primers (lane 2).

FIG. 10B is an agarose gel photograph to show the results of two stagePCR amplifications for Esx1 using EsxN1 (lane 1), EsxC2 (lane 2), a setof EsxN1-ACP and EsxC2 (lane 3), and a set of EsxN1-ACP and EsxC2-ACP(lane 4).

FIG. 10C is an agarose gel photograph to show the results of two stagePCR amplifications for Esx1 using a set of EsxN3 and EsxC5 (lanes 1 and2) and a set of EsxN3-ACP and EsxC5-ACP (lane 3).

FIG. 10D is an agrasoe gel photograph to show the results of non-stoptwo stage PCR amplifications for Esx1 using the primer EsxN1 (lane 1),EsxC2 (lane 2), a pair of EsxN1 and EsxC2 (lane 3) and a pair ofEsxN1-ACP and EsxC2-ACP (lane 4).

FIG. 11A is a photograph of agarose gels to show examples of the ACPused for detecting differentially expressed mRNAs during embryonicdevelopment using different stages of mouse conceptus tissues. The cDNAswere amplified using total RNA isolated from conceptus tissues at E4.5(lane 1), E11.5 (lane 2), and E18.5 (lane 3), with a set of ACP3 (SEQ IDNO. 3) and dT10-ACP1. The bands indicated by arrows represent the cDNAfragments amplified from differentially expressed mRNAs. The numbers ofthe arrows indicate the cDNA fragments used as probes in the Northernblot analysis of FIG. 13.

FIG. 11B is a photograph of agarose gels to show examples of the ACPused for detecting differentially expressed mRNAs during embryonicdevelopment using different stages of mouse conceptus tissues. The cDNAswere amplified using total RNA isolated from conceptus tissues at E4.5(lanes 1-2 and 7-8), E11.5 (lanes 3-4 and 9-10), and E18.5 (lanes 5-6and 11-12), with a set of ACP5 (SEQ ID NO. 5) and dTIO-ACP1 (the lanes16), and a set of ACP8 (SEQ ID NO. 8) and dT10-ACP1 (lanes 7-12),respectively. The bands indicated by arrows represent the cDNA fragmentsamplified from differentially expressed mRNAs. The numbers of the arrowsindicate the cDNA fragments used as probes in the Northern blot analysisof FIG. 13.

FIG. 11C is an agarose gel photograph to show the amplified cDNAproducts obtained from different stages of mouse conceptus samples(E4.5: lanes 1 and 2; E11.5: lanes 3 and 4; E18.5: lanes 5 and 6) usinga set of ACP 10 and dT10-ACP primers.

FIG. 11D is an agarose gel photograph to show the amplified cDNAproducts obtained from different stages of mouse conceptus samples(E4.5: lanes 1 and 2; E11.5: lanes 3 and 4; E18.5: lanes 5 and 6) usinga set of ACP14 and T10-ACP1 primers.

FIG. 12A is an agarose gel photograph to show the amplified cDNAproducts obtained from different stages of mouse conceptus samples(E4.5: lane 1; E11.5: lane 2; E18.5: lane 3) by one-stop two-stage PCRamplification using a set of ACP 10 and JYC5-T15-ACP primers.

FIG. 12B is an agarose gel photograph to show the amplified cDNAproducts obtained from different stages of mouse conceptus samples(E4.5: lane 1; E11.5: lane 2; E18.5: lane 3) by non-stop two-stage PCRamplification using a set of ACP10 and JYC5-T15-ACP primers.

FIG. 13 shows Northern blot analysis of six cDNA fragments amplifiedfrom differentially expressed mRNAs during embryonic development. Thesix 32P-labeled fragments indicated by arrows in FIG. 11 were used asprobes for Northern blot analysis. The arrows 1, 2, 3, 4, 5, and 6 areDEG1 (FIG. 13A), DEG3 (FIG. 13B), DEG2 (FIG. 13C), DEG8 (FIG. 13D), DEG5(FIG. 13E), and DEG7 (FIG. 13F), respectively, wherein the results ofthe DEG sequence analysis are shown in Table 1. DEG2 (SEQ ID NO. 31) andDEG5 (SEQ ID NO. 32) are turned out as novel genes (Table 2). Thecontrol panels (the lower part of each panel) show each gel beforeblotting, stained with ethidium bromide and photographed under UV light,demonstrating similar levels of 18S and 28S rRNA as a loading control.

FIG. 14 shows the expression patterns of a novel gene, DEG5, in a fullstage of mouse conceptus. Northern blot analysis was performed using theradio-labeled DEG5 cDNA fragment as a probe. Total RNA (20 μg/lane) wasprepared from mouse conceptuses at the gestation times as indicated. Thecontrol panel at the lower part shows a gel before blotting, stainedwith ethidium bromide and photographed under UV light, demonstratingsimilar levels of 18S and 28S rRNA as a loading control.

FIG. 15 is an agarose gel photograph to show the difference between theconventional 3′-RACE (lane 1) and the ACP-based 3′-RACE (lane 2) withregard to beta-actin 3′-RACE.

FIG. 16 is an agarose gel photograph to show the difference betweenCapFinder methods and ACP-based methods for mouse JunB (lanes 1 and 2)and beta-actin 5′-RACE (lanes 3 and 4) using the conventional primer(lanes 1 and 3) and ACP (lanes 2 and 4), respectively.

FIG. 17 is an agarose gel photograph to show the difference betweenCapFinder methods and ACP-based methods for mouse PLP-C alpha 5′-RACEusing the conventional primer (lane 1) and ACP (lanes 2, 3, and 4),respectively.

FIG. 18 shows the results of virtual Northern analysis by the CapFindermethods or ACP-based methods for the amplification of mouse full-lengthGAPDH cDNA.

FIG. 19 shows agarose gel photographs to show the results of genomicfingerprintings of 7 mouse stains using two different sets of arbitraryACPs.

FIG. 20 shows agarose gel photographs to show the amplified products ofmultiplex PCR by the conventional methods (A) or ACP-based methods (B)for the amplification of three target nucleic acids.

FIG. 21 shows agarose gel photographs to show the amplified products ofmultiplex PCR by the conventional methods (A) or ACP-based methods (Band C) for the amplification of four target nucleic acids. The ACP-basedmultiplex was conducted by one-stop (B) or non-stop (C) two-stage PCRamplification.

FIG. 22 shows an agarose gel photograph to show the results ofallele-specific amplification for a SNP in exon 4 of the human TP53 geneusing ACP.

FIG. 23 shows six agarose gel photographs which show the results ofallele-specific amplifications using ACPs for six additional SNPs eachpresent in different gene such as Beta-2 adrenergic receptor (ADRB2)(A), Chemokine (c-c motif) receptor 5 (CCR5) (B), Interleukin 13receptor (C), Leukocyte adhesion molecule-1 (LAM-1) (D), Tachykininreceptor 3 (TACR3) (E), and Interleukin 1, beta (IL1B) (F).

DETAILED DESCRIPTION OF THIS INVENTION

The present invention is generally directed to (a) an annealing controlprimer for the specificity of nucleic acid amplification and (b) itsapplications. The annealing control primer of this invention(hereinafter referred to as “ACP”) allows primer annealing to becontrolled in association with annealing temperature, such that thespecificity of nucleic acid amplification (in particular, PCR) can besignificantly improved. The principle of the ACP is based on thecomposition of an oligonucleotide primer having 3′- and 5′-ends distinctportions separated by at least one universal base or non-discriminatorybase. The present inventor has discovered that the universal base ornon-discriminatory base group positioned between the 3′- and 5′-endportions plays as a regulator in controlling primer annealing to atemplate nucleic acid in associated with annealing temperature duringnucleic acid amplification. The presence of universal base ornon-discriminatory base residue group positioned between the 3′- and5′-end portions interrupts the annealing of the 5′-end portion as wellas limits primer annealing to the 3′-end portion at certain annealingtemperature, which results in dramatic improvement of annealingspecificity. A universal base group positioned between the 3′- and5′-end portions of ACP is designed to define each portion. For thesereasons, the ACP is fundamentally different from the conventionalprimers in terms of the function for improving primer annealingspecificity under a particular stringency conditions during nucleic acidamplification.

The ACP of this invention is significantly effective and widelyaccessible to nucleic acid amplification-based applications. Also,various problems related to primer annealing specificity in theconventional PCR techniques can be fundamentally solved by the ACP. Themain benefits to be obtained from the use of the ACP during nucleic acidamplification (particularly PCR) are as follows:

(a) since the presence of an universal base residue group positionedbetween the 3′- and 5′-end portions restricts primer annealing portionto the 3′-end portion under such conditions that the 3′-end portionanneals to the template, the annealing sequence of a primer can beprecisely controlled, which make it possible to design a primer with adesired number of annealing sequence. It is particularly useful when anannealing portion of a primer has to be limited (e.g., single nucleotidepolymorphism (SNP) genotyping, DNA microarrary screening, and detectionof differentially expressed genes);

(b) since the presence of an universal base residue group positionedbetween the 3′- and 5′-end portions interrupts the annealing of the5′-end portion to the template under such conditions that the 3′-endportion anneals to the template, eventually the 5′-end portion notinvolved in the annealing provides the 3′-end portion with primerannealing specificity;

(c) the specificity of primer annealing is highly sensitive enough todetect even a single-base mismatching. Thus, it is particularly usefulfor the identification of a nucleotide variation in a target nucleicacid, including, for example, single nucleotide polymorphisms and pointmutations;

(d) ACP is capable of providing a primer with a high tolerance in“primer search parameters” for primer design such as primer length,annealing temperature, GC content, and PCR product length;

(e) ACP system provides two-stage PCR amplifications which allow theproducts to be excluded from non-specific amplification;

(f) the efficiency of PCR amplification is increased, which makes iteasier to detect rare mRNAs; and

(g) the reproducibility of PCR products is increased, which saves agreat amount of time and cost.

Principle of ACP

In one aspect of this invention, there is provided an annealing controlprimer for improving annealing specificity in nucleic acidamplification, which comprises: (a) a 3′-end portion having ahybridizing nucleotide sequence substantially complementary to a site ona template nucleic acid to hybridize therewith; (b) a 5′-end portionhaving a preselected arbitrary nucleotide sequence; and (c) a regulatorportion positioned between said 3′-end portion and said 5′-end portioncomprising at least one universal base or nondiscriminatory base analog,whereby said regulator portion is capable of regulating an annealingportion of said primer in association with annealing temperature.

The principle of ACP is based on the composition of an oligonucleotideprimer having 3′- and 5′-end distinct portions separated by a regulatorportion comprising at least one universal base or non-discriminatorybase and the effect of the regulator portion on the 3′- and 5′-endportions in the oligonucleotide primer. The presence of the regulatorportion comprising at least one universal base or non-discriminatorybase between the 3′- and 5′-end portions of ACP acts as a main factorwhich is responsible for the improvement of primer annealingspecificity.

The term “template” refers to nucleic acid. The term “nucleic acid” is adeoxyribonucleotide or ribonucleotide polymer in either single ordouble-stranded form, including known analogs of natural nucleotidesunless otherwise indicated. Therefore, the ACP of this invention can beemployed in nucleic acid amplification using single or double-strandedgDNA, cDNA or mRNA as template. The term “portion” used herein inconjunction with the primer of this invention refers to a nucleotidesequence separated by the regulator portion. The term “3′-end portion”or “5′-end portion” refers to a nucleotide sequence at the 3′-end or5′-end of the primer of this invention, respectively, which is separatedby the regulator portion.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally or produced synthetically, which is capable ofacting as a point of initiation of synthesis when placed underconditions in which synthesis of primer extension product which iscomplementary to a nucleic acid strand (template) is induced, i.e., inthe presence of nucleotides and an agent for polymerization such as DNApolymerase and at a suitable temperature and pH. The primer ispreferably single stranded for maximum efficiency in amplification.Preferably, the primer is an oligodeoxyribonucleotide. The primer ofthis invention can be comprised of naturally occurring dNMP (i.e., dAMP,dGM, dCMP and dTMP), modified nucleotide or non-natural nucleotide. Theprimer can also include ribonucleotides. The primer must be sufficientlylong to prime the synthesis of extension products in the presence of theagent for polymerization. The exact length of the primers will depend onmany factors, including temperature, application and source of primer.The term “annealing” or “priming” as used herein refers to theapposition of an oligodeoxynucleotide or nucleic acid to a templatenucleic acid, whereby said apposition enables the polymerase topolymerize nucleotides into a nucleic acid molecule which iscomplementary to the template nucleic acid or a portion thereof.

The 3′-end portion of ACP has a nucleotide sequence substantiallycomplementary to a site on a template nucleic acid molecule. The term“substantially complementary” in reference to primer is used herein tomean that the primer is sufficiently complementary to hybridizeselectively to a template nucleic acid sequence under the designatedannealing conditions, such that the annealed primer can be extended bypolymerase to form a complementary copy of the template. Therefore, thisterm has a different meaning from “perfectly complementary” or relatedterms thereof. It will be appreciated that the 3′-end portion of ACP canhave one or more mismatches to template to an extent that the ACP canserve as primer. Most preferably, the 3′-end portion of ACP has anucleotide sequence perfectly complementary to a site on a template,i.e., no mismatches.

The 3′-end portion of ACP may have a wide variety of nucleotidesequences depending on its applications as well as template sequence.For example, where the ACP is applied to the process involving reversetranscription such as differential display PCR, RACE, amplification offull-length cDNA, fingerprinting, identification of conserved homologysegment and the like, its 3′-end portion may have the nucleotidesequence which hybridizes to the polyadenosine (poly A) tail of an mRNA,preferably at least 8 deoxythymidine nucleotides, more preferably atleast 10 deoxythymidine nucleotides and the most preferably, at least 10contiguous deoxythymidine nucleotides. For the process involving reversetranscription as above, in one embodiment, the 3′-end portion of ACP hasat least 10 contiguous deoxythymidine nucleotides having 3′-V at its3′-end; in which V is one selected from the group consisting ofdeoxyadenosine, deoxycytidine and deoxyguanosine, in another embodiment,at least 10 contiguous deoxythymidine nucleotides having 3′-NV at its3′-end; in which V is one selected from the group consisting ofdeoxyadenosine, deoxycytidine and deoxyguanosine, and N is one selectedfrom the group consisting of deoxyadenosine, deoxythymidine,deoxycytidine and deoxyguanosine.

Furthermore, where the ACP is employed in amplification of a targetnucleic acid sequence, its 3′-end portion comprises a nucleotidesequence substantially complementary to a target sequence; indifferential display PCR, an arbitrary sequence substantiallycomplementary to a site in a cDNA from an mRNA; in RACE, a gene-specificsequence substantially complementary to a site in a cDNA from an mRNA;in amplification of 5′-enriched cDNAs, a random sequence of at least sixnucleotides substantially complementary to sites in mRNAs; inidentification of conserved homology segment, a nucleotide sequencesubstantially complementary to a consensus sequence found in a genefamily or degenerate sequence selected from a plurality of combinationsof nucleotides encoding a predetermined amino acid sequence; inidentification of a nucleotide variation (e.g., allelic site) in atarget nucleic acid, a nucleotide sequence comprising a nucleotidecomplementary to the corresponding nucleotide of a nucleotide variation;and in mutagenesis, a nucleotide sequence comprising at least onemismatch nucleotide to a target nucleic acid.

The term “arbitrary” nucleotide sequence is used herein to mean thenucleotide sequence that is chosen without knowledge of the sequence ofthe target nucleic acids to be amplified. The term arbitrary should notto be confused with “random” in reference to primer which connotes aprimer composed of a random population of primers each of different andrandom sequence. The term “degenerate” sequence in conjunction with ACPfor identification of conserved homology segment refers to thenucleotide sequence that is deducted from amino acid sequence, so thatthe degenerate sequence can form a pool of the nucleotide sequences fromone amino acid sequence due to degeneracy of genetic codon.

According to a preferred embodiment of the ACP, the pre-selectedarbitrary nucleotide sequence of the 5′-end portion is substantially notcomplementary to any site on the template nucleic acid.

According to a preferred embodiment, the annealing control primer ofthis invention can be represented by a general formula (1) of5′-Xp-Yq-Zr-3′, wherein Xp represents the 5′-end portion having thepre-selected arbitrary nucleotide sequence substantially notcomplementary to any site on the template nucleic acid; Yq representsthe regulator portion comprising at least one universal base ornon-discriminatory base analog; Zr represents the 3′-end portion havinga nucleotide sequence substantially complementary to a site on thetemplate nucleic acid; wherein p, q and r represent the number ofnucleotides; and wherein X, Y and Z is deoxyribonucleotide orribonucleotide.

The regulator portion comprising at least one universal base ornon-discriminatory base analog is responsible for the main function ofACP in associated with alteration of annealing temperature duringnucleic acid amplification. The term “universal base ornondiscriminatory base analog” used herein refers to one capable offorming base pairs with each of the natural DNA/RNA bases with littlediscrimination between them.

It has been widely known that nucleotides at some ambiguous positions ofdegenerate primers have been replaced by universal base or anon-discriminatory analogue such as deoxyinosine (Ohtsuka et al, 1985;Sakanari et al., 1989), 1-(2′-deoxy-beta-Dribofuranosyl)-3-nitropyrrole(Nichols et al., 1994) and 5-nitroindole (Loakes and Brown, 1994) forsolving the design problems associated with the degenerate primersbecause such universal bases are capable of non-specifically basepairing with all four conventional bases. However, there has not beenany report that this universal base or a nondiscriminatory analogue suchas deoxyinosine, 1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole and5-nitroindole is used to increase the specificity of primer annealingduring PCR.

The presence of universal base such as deoxyinosine,1-(2′-deoxy-beta-Dribofuranosyl)-3-nitropyrrole and 5-nitroindole in aprimer generates low annealing temperatures due to its weaker hydrogenbonding interactions in base pairing. As an extension of this theory,the present inventor has induced that the presence of the contiguousuniversal bases between the 3′-end and 5′-end of a primer could generatea region which has lower melting temperature, forms a boundary to eachof 3′- and 5′-end portions of the primer, and affect the annealing ofeach portion, respectively. This theory provides the basis of theannealing control primers of this invention.

In a preferred embodiment, the ACP contains at least 2 universal base ornon-discriminatory base analog residues between the 3′- and 5′-endportion sequences, more preferably, at least 3 universal bases ornon-discriminatory base analogs. Advantageously, the universal baseresidues between the 3′- and 5′-end portion sequences can be up to 15residues in length. According to one embodiment, the ACP contains 2-15universal base or non-discriminatory base analog residues. Mostpreferably, the universal bases between the 3′- and 5′-end portionsequences are about 5 residues in length.

With reference to the optimum number of universal base, i.e., 5residues, the minimum number of universal base residues between the 3′-and 5′-end portions of ACP is preferred in order to interrupt theannealing of the 5′-end portion to the template during nucleic acidamplification at certain annealing temperature. It is very likely thatthe length of universal base in the sequence (8-10 bases) does not makea significant difference on its own function in ACP.

The use of universal base residues between the 3′- and 5′-end portionsequences is considered as a key feature in the present inventionbecause it provides each portion (3′- and 5′-end) with a distinctannealing specificity in association with an annealing temperatureduring nucleic acid amplification, e.g. PCR.

According to a preferred embodiment, the universal base ornon-discriminatory base analog in the regulator portion includesdeoxyinosine, inosine, 7-deaza-2-2′-deoxyinosine, 2-aza-2′-deoxyinosine,2′-OMe inosine, 2′-F inosine, deoxy 3-nitropyrrole, 3-nitropyrrole,2′-OMe 3-nitropyrrole, 2′-F 3-nitropyrrole,1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, deoxy 5-nitroindole,5-nitroindole, 2′-OMe 5-nitroindole, 2′-F 5-nitroindole, deoxy4-nitrobenz imidazole, 4-nitrobenz imidazole, deoxy4-aminobenzimidazole, 4-aminobenzimidazole, deoxy nebularine, 2′-Fnebularine, 2′-F 4-nitrobenzimidazole, PNA5-introindole, PNA-nebularine,PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole,morpholino-5-nitroindole, morpholino-nebularine, morpholino-inosine,morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebularine,phosphoramidate-inosine, phosphoram idate-4-nitrobenzimidazole,phosphoramidate-3-nitropyrrole, 2′-0-methoxyethyl inosine,2′-0-methoxyethyl nebularine, 2′-0-methoxyethyl 5-nitroindole,2′-0-methoxyethyl 4-nitrobenzimidazole, 2′-0-methoxyethyl 3-nitropyrroleand combinations thereof, but not limited to. More preferably, theuniversal base or non-discriminatory base analog is deoxyinosine,1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole or 5-nitroindole, mostpreferably, deoxyinosine.

The preferred length of an oligonucleotide primer, as used herein, isdetermined from desired specificity of annealing and the number ofoligonucleotides having the desired specificity that are required tohybridize to the template. For example, an oligonucleotide primer of 20nucleotides is more specific than an oligonucleotide primer of 10nucleotides because the addition of each nucleotide to anoligonucleotide increases the annealing temperature of the primer to thetemplate.

The lengths of the 3′- and 5′-end portion sequences of the ACP may varyand depend in part on the objective of each application using ACP. In apreferred embodiment, the 3′-end portion of ACP is at least 6nucleotides in length, which is considered a minimal requirement oflength for primer annealing. More preferably, the 3′-end portionsequence is from 10 to 25 nucleotides and can be up to 60 nucleotides inlength. In another embodiment, the 3′-end portion of ACP can includeribonucleotides as well as deoxyribonucleotides.

In another preferred embodiment, the 5′-end portion of ACP contains atleast 15 nucleotides in length, which is considered a minimalrequirement of length for annealing under high stringent conditions.Preferably, the 5′-end portion sequence can be up to 60 nucleotides inlength. More preferably, the 5′-end portion sequence is from 6 to 50nucleotides, most preferably, from 20 to 25 nucleotides in length. Theentire ACP is preferably from 35 to 50 nucleotides in length, and can beup to 100 nucleotides in length.

The 5′-end portion of ACP has a pre-selected arbitrary nucleotidesequence substantially not complementary to any site on the templatenucleic acid and this nucleotide sequence can serves as a priming sitefor subsequent amplification. The term “pre-selected arbitrary”nucleotide sequence used herein refers as any defined or preselecteddeoxyribonucleotide, ribonucleotide, or mixed deoxyribonucleotidesequence which contains a particular sequence of natural or modifiednucleotides. In some embodiment, the pre-selected arbitrary nucleotidesequence of the 5′-end portion can be composed of a universal primersequence such as T3 promoter sequence, T7 promoter sequence, SP6promoter sequence, and M13 forward or reverse universal sequence. Usinga longer arbitrary sequence (about 25 to 60 bases) at the 5′-end portionof ACP reduces the efficiency of ACP, but shorter sequences (about 15 to17 bases) reduce the efficiency of annealing at high stringentconditions of ACP. It is also a key feature of the present invention touse a pre-selected arbitrary nucleotide sequence at the 5′-end portionof ACP as a priming site for subsequent amplification.

According to one embodiment of the present invention, some modificationsin the 5′-end portion of ACP can be made unless the modificationsabolish the advantages of the ACP, i.e., improvement in annealingspecificity. For example, the 5′-end portion can comprises a sequence orsequences recognized by a restriction endonuclease(s), which makes itfeasible to clone the amplified product into suitable vector. Inaddition, the 3-end portion can comprises at least one nucleotide with alabel for detection or isolation of amplified product. Suitable labelsinclude, but not limited to, fluorophores, chromophores,chemiluminescers, magnetic particles, radioisotopes, mass labels,electron dense particles, enzymes, cofactors, substrates for enzymes andhaptens having specific binding partners, e.g., an antibody,streptavidin, biotin, digoxigenin and chelating group. The 5′-endportion also comprises bacteriophage RNA polymerase promoter region.

According to the preferred embodiment of this invention, the ACP isapplied to PCR. More preferably, the PCR is performed under a first anda second annealing temperature, i.e., under different stringentconditions. The first annealing temperature may be equal to or lowerthan the second annealing temperature and preferably, the secondannealing temperature is higher than the first annealing temperature. Inthe PCR process performed under two different annealing temperatures,i.e., two-stage PCR, the 3′-end of ACP is involved in annealing at thefirst annealing temperature and the 5′-end of ACP incorporated intoamplified product of first amplification stage serves as a priming siteat the second annealing temperature. In this case, the advantages of ACPwill be demonstrated in accordance with the following assumptions:

(1) since a regulator portion of ACP is composed of at least oneuniversal base or non-discriminatory analogue which has lower Tm thanother portion in ACP due to its weaker hydrogen bonding interactions inbase pairing, the regulator portion of ACP is not favorable in annealingto the template nucleic acid under the conditions that the 3′-endportion of ACP anneals to a site of the template at a first annealingtemperature. Consequently, the presence of a regulator portioncomprising at least one universal base or non-discriminatory analoguebetween the 3′- and 5′-end portions of ACP restricts primer annealingportion to the 3′-end portion at first annealing temperature;

(2) the 5′-end portion which is not involved in the annealing under thefirst annealing temperature keeps bothering the annealing of the 3′-endportion to the template;

(3) thus, the strength in which the specific annealing of the 3′-endportion sequence occurs is relatively stronger than the strength inwhich non-specific annealing occurs, under the first annealingtemperature, which results in the improvement of primer annealingspecificity at the 3′-end portion;

(4) where the 5′-end portion comprises a pre-selected arbitrarynucleotide sequence, the portion serves as a priming site at a secondannealing temperature, which is high stringency conditions and alsoshould be higher than the first annealing temperature, for subsequentamplification of reaction product generated from annealing and extensionof the 3′-end portion sequence; and

(5) consequently, only the reaction product generated from annealing andextension of the 3′-end portion sequence can be amplified close to thetheoretical optimum of a two-fold increase of product for each PCR cycleunder the second annealing temperature.

Therefore, the 3′-end portion of ACP acts only as annealing site to thetemplate at the first annealing temperature and the 5′-end portion ofACP is used as a priming site at the second annealing temperature forthe subsequent amplification of the product generated by contacting andextending the 3′-end portion of ACP to the template.

It may be appreciated that the ACP of the present invention is veryuseful in a variety of primer-based nucleic acid amplification methodsincluding the methods of Miller, H. I. (WO 89/06700) and Davey, C. etal. (EP 329,822), Ligase Chain Reaction (LCR, Wu, D. Y. et al., Genomics4:560 (1989)), Polymerease Ligase Chain Reaction (Barany, PCR Methodsand Applic., 1:5-16 (1991)), Gap-LCR (WO 90/01069), Repair ChainReaction (EP 439,182), 3SR (Kwoh et al., PNAS, USA, 86:1173 (1989)) andNASBA (U.S. Pat. No. 5,130,238), but not limited to.

In another aspect of this invention, there is provided a kit comprisingthe annealing control primer or the annealing control primer setaccording to the present invention. According to one embodiment of thisinvention, this kit further comprises a primer or a primer pair having anucleotide sequence corresponding to the 5′-end portion of the ACP; incase that the 5′-end portion comprises universal primer sequence, it ismore preferred that the kit comprises the universal primers. The presentkits may optionally include the reagents required for performing PCRreactions such as buffers, DNA polymerase, DNA polymerase cofactors, anddeoxyribonucleotide-5′-triphosphates. Optionally, the kits may alsoinclude various polynucleotide molecules, reverse transcriptase, variousbuffers and reagents, and antibodies that inhibit DNA polymeraseactivity. The kits may also include reagents necessary for performingpositive and negative control reactions. Optimal amounts of reagents tobe used in a given reaction can be readily determined by the skilledartisan having the benefit of the current disclosure. The kits,typically, are adapted to contain in separate packaging or compartmentsthe constituents afore-described.

The ACP of the subject invention can be applied to a variety of nucleicacid amplification-based technologies. Representative examples to provethe effect of ACP are:

I. Application to amplifying a nucleic acid sequence;

II. Application to amplifying a target nucleic acid sequence;

III. Application to multiplex DNA amplification;

IV. Application to the identification of differentially expressed genes;

V. Application to rapid amplification of cDNA ends (RACE);

VI. Application to amplifying full-length cDNA;

VII. Application to amplifying 5′-enriched cDNA;

VIII. Application to DNA or RNA fingerprinting;

IX. Application to the identification of conserved homology segments inmultigene families;

X. Application to identification of a nucleotide sequence variation;

XI. Application to mutagenesis; and

XII. Other applications.

I. Application to Amplifying a (target) Nucleic Acid Sequence

In still another of this invention, there is provided a method foramplifying a nucleic acid sequence from a DNA or a mixture of nucleicacids, comprising performing an amplification reaction using primers,characterized in that at least one primer is derived from any one of ACPdescribed above. Preferably, the primer according to the structure ofACP is one having at its 3′ end portion a hybridizing sequencesubstantially complementary to a region of the nucleic acid sequence tohybridize therewith.

In a specific embodiment of this method, there is provided a methodusing two stage amplifications for amplifying a nucleic acid sequencefrom a DNA or a mixture of nucleic acids, which comprises:

(a) performing a first-stage amplification of the nucleic acid sequenceat a first annealing temperature comprising at least two cycles ofprimer annealing, primer extending and denaturing, using the primer pairof any one of the ACP described above each having at its 3′ end portiona hybridizing sequence substantially complementary to a region of thenucleic acid sequence to hybridize therewith, under conditions in whicheach primer anneals to the region of the nucleic acid sequence, wherebythe amplification product of the nucleic acid sequence is generated; and

(b) performing a second-stage amplification of the amplification productgenerated from step (a) at a second annealing temperature, which is highstringent conditions, comprising at least one cycle of primer annealing,primer extending and denaturing, using the same primers as used in step(a) or a primer pair each comprising a pre-selected arbitrary nucleotidesequence corresponding to each 5′-end portion of the primers used instep (a), under conditions in which each primer anneals to the 3′- and5′-ends of the amplification product, respectively, whereby theamplification product is re-amplified.

Where the method is applied to the amplification of a target nucleicacid sequence, the primer pair used has at its 3′-end portion ahybridizing sequence substantially complementary to a region of thetarget nucleic acid sequence to hybridize therewith. Therefore, in afurther aspect of this invention, there is provided a method forselectively amplifying a target nucleic acid sequence from a DNA or amixture of nucleic acids, wherein the method comprises performing anamplification reaction using primers, characterized in that at least oneprimer is derived from the ACP described above. Preferably, the primeraccording to the structure of ACP is one having at its 3′end portion ahybridizing sequence substantially complementary to a region of thetarget nucleic acid sequence to hybridize therewith.

In a specific embodiment of this method, there is provided a methodusing two stage amplifications for selectively amplifying a targetnucleic acid sequence from a DNA or a mixture of nucleic acids, whichcomprises:

performing a first-stage amplification of the target nucleic acidsequence at a first annealing temperature comprising at least two cyclesof primer annealing, primer extending and denaturing, using the primerpair of any one of the ACP described above each having at its 3′ endportion a hybridizing sequence substantially complementary to a regionof the target nucleic acid sequence to hybridize therewith, underconditions in which each primer anneals to its target nucleotidesequence, whereby the amplification product of the target nucleotidesequence is generated; and

performing a second-stage amplification of the amplification productgenerated from step (a) at a second annealing temperature, which is highstringent conditions, comprising at least one cycle of primer annealing,primer extending and denaturing, using the same primers as used in step(a) or a primer pair each comprising a pre-selected arbitrary nucleotidesequence corresponding to each 5′-end portion of the primers used instep (a), under conditions in which each primer anneals to the 3′- and5′-ends of the amplification product, respectively, whereby theamplification product is re-amplified.

Where the template for amplification is mRNA, the production of cDNA isrequired prior to amplification. Therefore, in still further aspect ofthis invention, there is provided a method for selectively amplifying atarget nucleic acid sequence from an mRNA, wherein the method comprisesreverse transcribing the mRNA and performing an amplification reactionusing primers, characterized in that at least one primer is derived fromthe ACP described above. Preferably, the primer according to thestructure of ACP is one having at its 3′ end portion a hybridizingsequence substantially complementary to a region of the target nucleicacid sequence to hybridize therewith.

In a specific embodiment of this invention, there is provided a methodusing two stage amplifications for selectively amplifying a targetnucleic acid sequence from an mRNA which comprises:

contacting the mRNA with an oligonucleotide dT primer which ishybridized to polyA tail of the mRNA under conditions sufficient fortemplate driven enzymatic deoxyribonucleic acid synthesis to occur;

reverse transcribing the mRNA to which the oligonucloetide dT pirmerhybridizes to produce a first DNA strand that is complementary to themRNA to which the oligonucloetide dT pirmer hybridizes;

performing a first-stage amplification of the target nucleic acidsequence from the first DNA strand obtained from step (b) at a firstannealing temperature comprising at least two cycles of primerannealing, primer extending and denaturing, using the primer pair of ACPdescribed above having at its 3′ end portion a hybridizing sequencesubstantially complementary to a region of the target nucleic acidsequence to hybridize therewith, under conditions in which each primeranneals to its target nucleotide sequence, whereby the amplificationproduct of the target nucleotide sequence is generated; and

performing a second-stage amplification of the amplification productgenerated from step (c) at a second annealing temperature, which is highstringent conditions, comprising at least one cycle of primer annealing,primer extending and denaturing, using the same primers as used in step(c) or a primer pair each comprising a pre-selected arbitrary nucleotidesequence corresponding to each 5′-end portion of the primers used instep (c), under conditions in which each primer anneals to the 3′- and5′-ends of the amplification product, respectively, whereby theamplification product is re-amplified.

Since the amplification methods of this invention employs the ACP ofthis invention, the common descriptions between them are omitted inorder to avoid the complexity of this specification leading to unduemultiplicity.

This application using ACP of the subject invention can provide animproved method for selectively amplifying a target nucleic acidsequence from a nucleic acid or a mixture of nucleic acids (DNA or mRNA)by performing nucleic acid amplifications, preferably, PCR. Since theeffect of ACP provides the conventional primers with primer annealingspecificity regardless of “primer search parameters” for primer designsuch as primer length, annealing temperature, GC content and productlength, it is particularly recommended to use the ACP when theconventional primers used to amplify a target nucleic acid fragment aretoo sensitive to such parameters to generate specific nucleic acidamplification products.

A schematic representation for selectively amplifying a target nucleicacid of double-stranded DNA using novel ACP system as described above isillustrated in FIG. 1A. FIG. 1B illustrates a schematic representationfor selectively amplifying a target nucleic acid of mRNA using novel ACPsystem. Referring to FIGS. 1A and 1B, the present methods will bedescribed in more detail.

The present methods for amplifying a nucleic acid sequence may becarried out in accordance with various primer-based nucleic acidamplifications known in the art. Preferably, the methods are carried outaccording to the two stage amplifications developed by the presentinventor, more preferably, the amplification is performed by polymerasechain reaction known in the art and most preferably, hot start PCRmethod.

The methods of the present invention, for amplifying a nucleic acidsequence can be used to amplify any desired nucleic acid molecule. Suchmolecules may be either DNA or RNA. The molecule may be in either adouble-stranded or single-stranded form, preferably, double-stranded.Where the nucleic acid as starting material is double-stranded, it ispreferred to render the two strands into a single-stranded, or partiallysingle-stranded, form. Methods known to separate strands includes, butnot limited to, heating, alkali, formamide, urea and glycoxal treatment,enzymatic methods (e.g., helicase action) and binding proteins. Forinstance, strand separation can be achieved by heating at temperatureranging from 80° C. to 105° C. General methods for accomplishing thistreatment are provided by Joseph Sambrook, et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001).

Where a mRNA is employed as starting material for amplification, areverse transcription step is necessary prior to amplification, detailsof which are found in Joseph Sambrook, et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001); and Noonan, K. F. et al., Nucleic Acids Res.16:10366 (1988)). For reverse transcription, an oligonucleotide dTprimer hybridizable to poly A tail of mRNA is used. The oligonucleotidedT primer is comprised of dTMPs, one or more of which may be replacedwith other dNMPs so long as the dT primer can serve as primer. Reversetranscription can be done with a reverse transcriptase that has RNase Hactivity. If one uses an enzyme having RNase H activity, it may bepossible to omit a separate RNase H digestion step, by carefullychoosing the reaction conditions.

The present methods do not require that the molecules to be amplifiedhave any particular sequence or length. In particular, the moleculeswhich may be amplified include any naturally occurring procaryotic,eukaryotic (for example, protozoans and parasites, fungi, yeast, higherplants, lower and higher animals, including mammals and humans) or viral(for example, Herpes viruses, HIV, influenza virus, Epstein-Barr virus,hepatitis virus, polio virus, etc.) or viroid nucleic acid. The nucleicacid molecule can also be any nucleic acid molecule which has been orcan be chemically synthesized. Thus, the nucleic acid sequence may ormay not be found in nature.

The ACP used for the present invention is hybridized or annealed to aregion on template so that double-stranded structure is formed.Conditions of nucleic acid hybridization suitable for forming suchdouble stranded structures are described by Joseph Sambrook, et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2001) and Haymes, B. D., et al.,Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C. (1985). The sequence of the 3′-end portion of ACP needs not toexhibit precise complementarity, but need only to be substantiallycomplementary in sequence to be able to form a stable double-strandedstructure. Thus, departures from complete complementarity arepermissible, so long as such departures are not sufficient to completelypreclude hybridization to form a double-stranded structure.Hybridization of ACP to a region on template nucleic acid is aprerequisite for its template-dependent polymerization with polymerases.Factors (see Joseph Sambrook, et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); and Haymes, B. D., et. al., Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, D.C. (1985)) which affect thebase pairing of ACP to its complementary nucleic acids subsequentlyaffect priming efficiency. The nucleotide composition of ACP can affectthe temperature at which annealing is optimal and therefore can affectits priming efficiency.

A variety of DNA polymerases can be used in the amplification step ofthe present methods, which includes “Klenow” fragment of E. coli DNApolymerase 1, a thermostable DNA polymerase and bacteriophage T7 DNApolymerase. Preferably, the polymerase is a thermostable DNA polymerasesuch as may be obtained from a variety of bacterial species, includingThermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis,Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu).Many of these polymerases may be isolated from bacterium itself orobtained commercially. Polymerase to be used with the subject inventioncan also be obtained from cells which express high levels of the clonedgenes encoding the polymerase. When a polymerization reaction, is beingconducted, it is preferable to provide the components required for suchreaction in excess in the reaction vessel. Excess in reference tocomponents of the amplification reaction refers to an amount of eachcomponent such that the ability to achieve the desired amplification isnot substantially limited by the concentration of that component. It isdesirable to provide to the reaction mixture an amount of requiredcofactors such as Mg2+, and dATP, dCTP, dGTP and dTTP in sufficientquantity to support the degree of amplification desired.

All of the enzymes used in this amplification reaction may be activeunder the same reaction conditions. Indeed, buffers exist in which allenzymes are near their optimal reaction conditions. Therefore, theamplification process of the present invention can be done in a singlereaction volume without any change of conditions such as addition ofreactants.

It would be understood that the 5′-end portions of a set of ACPs used inthe step of the first-stage amplification could comprise identical ordifferent sequences; if they are identical, one primer corresponding tothe sequence of 5′-end portion will be used in the step of thesecond-stage amplification, whereas if they are different, two primerseach corresponding to the sequence of each 5′-end portion of ACPs willbe used in the step of the second-stage amplification.

The present invention includes an alternative process for selectivelyamplifying a target nucleic acid fragment from a nucleic acid or amixture using ACP, wherein a set of primers comprising an ACP and aconventional primer can be used in the first amplification step, insteadof a set of ACP. The term “conventional primer” used herein refers toany primer having a structure different from ACP, especially; in termsof the presence of the regulator portion containing universal base. Inthis case, the conventional primer is added only the first amplificationstep with the ACP and only one pre-selected arbitrary primercorresponding to the 5′-end portion sequence of the ACP is added in thesecond amplification step. In preferred embodiment, the alternativeprocess can be used when each 3′-portion of a pair of ACP to be used inthe first amplification step has different melting temperature (Tm).“Tm” refers to the temperature at which half the primers are annealed tothe target region.

Two amplification steps of the present methods (in case of amplificationfrom mRNA, including reverse transcritptation) are separated only intime. The first-stage amplification should be followed by thesecond-stage amplification. It would be understood that the first-stageamplification reaction mixture could include the primers correspondingto the 5′-end portion which will be used to anneal to the sequences ofthe 5′-end portions of the ACPs in the second-stage amplification, whichmeans that the primers corresponding to the 5′-end portion can be addedto the reaction mixture at the time of or after the first-stageamplification step.

As an alternative process, in the second-stage amplification step thecomplete sequences of the ACPs used in the first-stage amplificationstep, instead of the primers corresponding to the 5′-end portions of theACPs, can be used as primers at the high stringent conditions forre-amplifying the product generated from the first-stage amplificationstep, wherein the 3′- and 5′-ends of the product from the firstamplification step which is generated from annealing and extension ofthe 3′-end portion sequence of the set of ACP to the template nucleicacid at the low stringent conditions comprise the sequence orcomplementary sequence of ACP and also serve as perfect paring sites tothe set of ACP. In this view, this alternative process is preferredbecause this need not further add the primers corresponding to the5′-end portions of the ACPs to the reaction mixture at the time of orafter the first-stage amplification step. FIG. 1A also illustrates aschematic representation for selectively amplifying a target nucleicacid by the alternative process stated above.

Annealing or hybridization in the present methods is performed understringent conditions that allow for specific binding between anucleotide sequence and ACP. Such stringent conditions for annealingwill be sequence-dependent and varied depending on environmentalparameters. In the present methods, the second-stage amplification isgenerally performed under higher stringent conditions than thefirst-stage amplification.

In a preferred embodiment, the first annealing temperature ranges fromabout 30° C. to 68° C. for the first-stage amplification step, morepreferably, 40° C. to 65° C. It is preferred that the second annealingtemperature ranges from about 50° C. to 72° C. for the second-stageamplification. According to a more preferred embodiment, the firstannealing temperature is equal to or lower than the second annealingtemperature. The length or melting temperature (Tm) of the 3′-endportion sequence of ACP will determine the annealing temperature for thefirst-stage amplification. For example, in case that ACP comprises 10arbitrary nucleotides at the 3′-end portion, preferably, the annealingtemperature will be about between 45° C. and 55° C. for the first-stageamplification.

According to the present methods, the first-stage amplification underlow stringent conditions is carried out for at least 2 cycles ofannealing, extending and denaturing to improve the specificity of primerannealing during the first-stage amplification, and through thesubsequent cycles, the second-stage amplification is processed moreeffectively under high stringent conditions. The first-stageamplification can be carried out up to 30 cycles. In a preferredembodiment, the first-stage amplification is carried out for 2 cycles.In another embodiment, the second-stage amplification under highstringent conditions is carried out for at least one cycle (preferably,at least 5 cycles) and up to 45 cycles to amplify the first-stageproduct. In a more preferred embodiment, the second-stage amplificationis carried out for 25-35 cycles. High and low stringent conditions maybe readily determined from the standard known in the art. “Cycle” refersto the process which results in the production of a copy of targetnucleic acid. A cycle includes a denaturing step, an annealing step, andan extending step.

In the most preferable embodiment, the amplification is performed inaccordance with PCR which is disclosed in U.S. Pat. Nos. 4,683,195,4,683,202, and 4,800,159.

According to a preferred embodiment, when the first-stage amplificationis carried out, the 3′-end portion of the primer pair of ACP is involvedin annealing at the first annealing temperature and when thesecond-stage amplification is carried out, the 5′-end portion of theprimer pair serves as a priming site. Such alteration of the portion toinvolve in annealing is mainly ascribed to the ACP itself, inparticular, the regulator portion of ACP. In the present methods, theregulator portion of ACP is capable of restricting the annealing portionof ACP to its 3′-end portion at the first annealing temperature,responsible for improving annealing specificity to a target sequence.

The present methods may be combined with many other processes known inthe art to achieve a specific aim. For example, the isolation (orpurification) of amplified product may follow the second-stageamplification. This can be accomplished by gel electrophoresis, columnchromatography, affinity chromatography or hybridization. In addition,the amplified product of this invention may be inserted into suitablevehicle for cloning. Furthermore, the amplified product of thisinvention may be expressed in suitable host harboring expression vector.In order to express the amplified product, one would prepare anexpression vector that carries the amplified product under the controlof, or operatively linked to a promter. The promoter is originated fromthe vector itselt or the end portion of the amplified product, which maycorrespond to 5′-end portion of the ACP. Many standard techniques areavailable to construct expression vectors containing the amplifiedproduct and transcriptional/translational/control sequences in order toachieve protein or peptide expression in a variety of host-expressionsystems. The promoter used for prokaryotic host includes, but notlimited to, pLλ promoter, trp promoter, lac promoter and T7 promoter.The promoter used for eukaryotic host includes, but not limited to,metallothionein promoter, adenovirus late promoter, vaccinia virus 7.5Kpromoter and the promoters derived from polyoma, adenovirus 2, simianvirus 40 and cytomegalo virus. Certain examples of prokaryotic hosts areE. coli, Bacillus subtilis, and other enterobacteriaceae such asSalmonella typhimurium, Serratia marcescens, and various Pseudomonasspecies. In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. In addition to mammalian cells, these include insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus); and plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, tobacco mosaicvirus) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing one or more coding sequences. The expressedpolypeptide from the amplified product may be generally purified with avariety of purposes in accordance with the method known in the art.

In another aspect of this invention, there is provided a kit for nucleicacid amplification of the instant invention described previously, whichcomprises the annealing control primer or annealing control primer setindicated above.

In still another aspect of this invention, there is provided a kit forselective amplification of a target nucleic acid sequence from DNAdescribed previously, which comprises the annealing control primer orannealing control primer set indicated above.

In further aspect of this invention, there is provided a kit forselective amplification of a target nucleic acid sequence from mRNAdescribed previously, which comprises the annealing control primer orannealing control primer set indicated above.

According to one embodiment of this invention, these kits furthercomprises a primer or a primer pair each having a nucleotide sequencecorresponding to the 5′-end portion of the ACP; in case that the 5′-endportion comprises universal primer sequence, it is more preferred thatthe kit comprises the universal primers. The present kits may optionallyinclude the reagents required for performing PCR reactions such asbuffers, DNA polymerase, DNA polymerase cofactors, anddeoxyribonucleotide-5′-triphosphates. Optionally, the kits may alsoinclude various polynucleotide molecules, reverse transcriptase, variousbuffers and reagents, and antibodies that inhibit DNA polymeraseactivity. The kits may also include reagents necessary for performingpositive and negative control reactions. Optimal amounts of reagents tobe used in a given reaction can be readily determined by the skilledartisan having the benefit of the current disclosure. The kits,typically, are adapted to contain in separate packaging or compartmentsthe constituents afore-described.

II. Application to Multiplex DNA Amplification

This application using ACP of the subject invention can also provide animproved method for amplifying more than one target sequence using morethan one pair of primers in the same reaction. In general, it isextremely difficult to set up PCR conditions to amplify more than 10targets in parallel because an optimal PCR reaction is required toamplify even one specific locus without any unspecific by-products, sothat those researchers who have achieved multiplex PCR have had to workhard to optimize their systems. Since annealing needs to take place at asufficiently high temperature to allow the perfect DNA-DNA matches tooccur in the reaction, the ACP of the subject invention is ideal in theoptimization of multiplex DNA amplification due to its function ofimproving the specificity of amplification. “Multiplex PCR” as usedherein refers to the simultaneous amplification of multiplex DNA targetsin a single polymerase chain reaction (PCR) mixture.

In still further aspect of this invention, there is provided a methodfor amplifying more than one target nucleotide sequence simultaneouslyusing more than one pair of primers in the same reaction, wherein themethod comprises performing an amplification reaction using primers,characterized in that at least one primer is derived from any one of ACPdescribed above. Preferably, the primer according to the structure ofACP is one having at its 3′ end portion a hybridizing sequencesubstantially complementary to a region of the target nucleic acidsequence to hybridize therewith.

In a specific embodiment of this invention, there is provided the methodusing two stage amplifications, which comprises:

(a) performing a first-stage amplification of more than one targetnucleotide sequence at a first annealing temperature comprising at leasttwo cycles of primer annealing, primer extending and denaturing, usingthe primer pairs of any one of ACP above in which its 3′ end portioneach of the primer pairs has a hybridizing nucleotide sequencesubstantially complementary to a region of the target nucleic acidsequence to hybridize therewith, under conditions in which each of eachprimer pair anneals to its target nucleotide sequence, whereby theamplification products of target nucleotide sequences are generated; and

(b) performing a second-stage amplification of the amplificationproducts generated from step (a) at a second annealing temperature,which is high stringent conditions, comprising at least one cycle ofprimer annealing, primer extending and denaturing, using the same primerpairs as used in step (a) or primer pairs each comprising a pre-selectedarbitrary nucleotide sequence corresponding to each 5′-end portion ofthe primer pairs used in step (a), under conditions in which each ofeach primer pair anneals to the 3′- and 5′-end sequences of theamplification products generated from step (a), respectively, wherebythe amplification products are re-amplified in the same reaction.

Since this application using the ACP of this invention is carried out inaccordance with the present methods for amplification of nucleic acidsequence previously discussed, except for using more than one targetnucleotide sequence and primer pairs, the common descriptions betweenthem are omitted in order to avoid the complexity of this specificationleading to undue multiplicity.

For instance, the composition and structure of ACP used and theconditions for amplification, are common between this process and thepresent methods for amplification of nucleic acid sequence previouslydiscussed.

In a preferred embodiment, the amplified products from each of targetnucleotide sequences are different in size for subsequent analysis.

According to a preferred embodiment, the amplification products ofmultiplex target nucleotide sequences may be analyzed through sizeseparation. The size separation comparison is performed using a varietyof method known in the art, such as electrophoresis through apolyacrylamide gel matrix or agarose gel matrix and nucleotidesequencing. The nucleotide sequencing may be rapidly carried out with anautomatic sequencer available from various manufacturor.

As exemplified in Example below, the ACP of this invention permits thefinal amplified products to be free from the background problems as wellas non-specificity arising from the conventional primers used inmultiplex nucleic acid amplification methods known in the art.

The advantage of the multiplex amplification is that numerous diseasesor specific nucleotide sequence alterations (e.g., single nucleotidepolymorphism or point mutation) can be assayed in the same reaction.

The number of analyses that can be run simultaneously is unlimited;however, the upper limit is probably about 20 and is likely to bedependent on the size difference required for resolution and methodsthat are available to resolve the amplified product.

In another aspect of this invention, there is provided a kit foramplifying more than one target nucleotide sequence simultaneously inthe same reaction, which comprises the annealing control primer orannealing control primer set described above. According to oneembodiment of this invention, these kits further comprises a primer or aprimer pair having a nucleotide sequence corresponding to the 5′-endportion of the ACP; in case that the 5′-end portion comprises universalprimer sequence, it is more preferred that the kit comprises theuniversal primers. The present kits may optionally include the reagentsrequired for performing PCR reactions such as buffers, DNA polymerase,DNA polymerase cofactors, and deoxyribonucleotide-5′-triphosphates.Optionally, the kits may also include various polynucleotide molecules,reverse transcriptase, various buffers and reagents, and antibodies thatinhibit DNA polymerase activity. The kits may also include reagentsnecessary for performing positive and negative control reactions.Optimal amounts of reagents to be used in a given reaction can bereadily determined by the skilled artisan having the benefit of thecurrent disclosure. The kits, typically, are adapted to contain inseparate packaging or compartments the constituents afore-described.

The method and kit of the present invention may be applied to thediagnosis of genetic and infectious diseases, gender determination,genetic linkage analysis, and forensic studies.

III. Application to Identification of Differentially Expressed Genes

This application using ACP of the subject invention can also provide animproved method for detecting and cloning cDNAs complementary todifferentially expressed mRNAs in two or more nucleic acid samples.

In still further aspect of this invention, there is provided a methodfor detecting DNA complementary to differentially expressed mRNA in twoor more nucleic acid samples, wherein the method comprises reversetranscribing the mRNA and performing an amplification reaction usingprimers, characterized in that at least one primer is derived from anyone of ACP described above. Preferably, the primer according to thestructure of ACP is one having at its 3′ end portion a hybridizingsequence (more preferably, arbitrary sequence) substantiallycomplementary to a region of cDNA strands generated from reversetranscription.

In a specific embodiment of this invention, there is provided the methodusing two stage amplifications, which comprises:

(a) providing a first sample of nucleic acids representing a firstpopulation of mRNA transcripts and a second sample of nucleic acidsrepresenting a second population of mRNA transcripts;

(b) separately contacting each of the first nucleic acid sample and thesecond nucleic acid sample with a first primer of any one of ACPdescribed above, in which the 3′-end portion of the first primercomprises a hybridizing nucleotide sequence substantially complementaryto a first site in the differentially expressed mRNA to hybridizetherewith, under conditions sufficient for template driven enzymaticdeoxyribonucleic acid synthesis to occur;

(c) reverse transcribing the differentially expressed mRNA to which thefirst primer hybridizes to produce a first population of first cDNAstrands that are complementary to the differentially expressed mRNA inthe first nucleic acid sample to which the first primer hybridizes, anda second population of first cDNA strands that are complementary to thedifferentially expressed mRNA in the second nucleic acid sample to whichthe first primer hybridizes;

(d) purifying and quantifying each of the first and second populationsof first cDNA strands;

(e) performing a first-stage amplification of each of the first andsecond population of first DNA strands obtained from step (d) at a firstannealing temperature comprising at least one cycle of primer annealing,primer extending and denaturing, using a second primer of any one of ACPdescribed above having at its 3′ end portion a hybridizing sequencesubstantially complementary to a second site in the first and secondpopulations of first cDNA strands, under conditions in which the secondprimer anneals to the second site in each population of the first cDNAstrands, whereby first and second populations of second cDNA strands aregenerated;

(f) performing a second-stage amplification of each second cDNA strandgenerated from step (e) at a second annealing temperature, which is highstringent conditions, comprising at least two cycles of primerannealing, primer extending and denaturing, using the same first andsecond primers as used in steps (b) and (e), respectively, or a primerpair each comprising a pre-selected arbitrary nucleotide sequencecorresponding to each 5′-end portion of the first and second primersused in steps (b) and (e), respectively, under conditions in which eachprimer anneals to the 3′- and 5′-end sequences of each second cDNAstrand, respectively, whereby amplification products of the second cDNAstrands are generated, and

(g) comparing the presence or level of individual amplification productsin the first and second populations of amplification products obtainedfrom step (f).

Since this application using the ACP of this invention employs thepresent methods for amplification of nucleic acid sequence previouslydiscussed, the common descriptions between them are omitted in order toavoid the complexity of this specification leading to unduemultiplicity.

A schematic representation for identifying differentially expressedgenes using novel ACP is illustrated in FIG. 2A.

In the present method, the nucleic acid sample representing a populationof mRNA transcripts can be obtained from a wide variety of biologicalmaterials. In general, the first nucleic acid sample comprises mRNAexpressed in a first cell and the second nucleic acid sample comprisesmRNA expressed in a second cell. In particular, the first nucleic acidsample comprises mRNA expressed in a cell at a first developmental stageand the second nucleic acid sample comprises mRNA expressed in a cell ata second developmental stage. In addition, the first nucleic acid samplecomprises mRNA expressed in a tumorigenic cell and the second nucleicacid sample comprises mRNA expressed in a normal cell.

Steps (e) and (f) of the subject application may occur in a single tubeusing the same reaction mixture except for primers, which means thatsteps (e) and (f) are separated only in time. It would be understoodthat the primers corresponding to the 5′-end portion could be added tothe reaction mixture at the time of or after the second cDNA strandsynthesis. In a preferred embodiment, the primers corresponding to the5′-end portion are added to the reaction mixture right after step (e) iscompleted, followed by subsequent PCR amplification of second cDNAstrands.

It would be also understood that the 5′-end portion sequences of thefirst and second ACPs used in steps (b) and (e), respectively, could beidentical or different sequences; if they are identical, one primercorresponding to the sequence of 5′-end portion will be used in the step(f), whereas if they are different, two primers each corresponding tothe sequence of each 5′-end portion of ACPs will be used in the step(f). In a preferred embodiment, the 5′-end portion sequences of thefirst and second ACPs used in steps (b) and (e) are different and thus,two primers each corresponding to the sequence of each 5′-end portion ofACPs are used in step (f).

As an alternative process, in step (f) the complete sequences of thefirst and second ACPs used in steps (b) and (e), respectively, insteadof the primers corresponding to the 5′-end portions of the ACPs, can beused as primers at the high stringent conditions for amplifying eachsecond DNA strand obtained from step (e), wherein the 3′- and 5′-ends ofthe second DNA strands which are initially synthesized using the secondACP comprise the sequence of the first ACP and the complementarysequence of the second ACP, respectively, and also serve as perfectpairing sites to the first and second ACPs. In this view, thisalternative process is preferred because there is no need to add theprimers corresponding to the 5′-end portions of the ACPs to the reactionmixture at the time of or after first-stage PCR reaction. FIG. 2Billustrates a schematic representation for identifying differentiallyexpressed genes by the alternative process stated above.

The method of the subject application for detecting differences in geneexpression uses only a single cDNA synthesis primer (the first ACP) toreact with mRNA, unlike conventional Differential Display PCR whichrequires multiple cDNA synthesis anchor primers. In the originaldifferential display method outlined by Liang and Pardee in 1992, twelveanchor primers have been introduced. The anchor primers for example,having a sequence of T12 MN, where M is A, C, or G and N is A, C, G orT, produced twelve separate cDNA populations. Recently, modified anchorprimers have been proposed by altering the number of nucleotides such asone or three instead of two at the 3′-end which can hybridize to asequence that is immediately 5′ to the poly A tail of mRNAs or byextending additional nucleotides at the 5′-end while retaining the Oligo(dT)9-12 MN tail resulting in at least 21 nucleotides in length(Villeponteau et al., 1996, Combates et al., 2000).

The subject invention concerns the embodiments of the ACP used in thismethod for the identification of differentially expressed genes, whereinthe first ACP used in step (b) is represented by the following generalformula (2): 5′-dXp-dYq-dTr-3′ wherein dX is one of the fourdeoxyribonucleotides, A, C, G, or T; dY is a regulator portioncomprising universal bases responsible for the main function of the ACPassociated with alteration of annealing temperature during PCR; dT is aT deoxyribonucleotide; p, q, and r represent an integer, respectively;dXp represents the 5′-end portion and contains a pre-selected arbitrarynucleotide sequence; dYq contains at least 2 universal bases; dTrrepresents the 3′-end portion; the nucleotide sequence of the 3′-endportion should have lower Tm than that of the 5′-end portion. Theformula (2) basically follows the rule of formula (1). The 3′-endportion of formula (2) consists of the sequences capable of annealing tothe poly A tail of mRNA and serves as a cDNA synthesis primer forreverse transcription of mRNA.

In a preferred embodiment, the 3′-end portion of the first ACP used instep (b) contains at least 6 T nucleotides in length, which isconsidered a minimal requirement of length for primer annealing. Morepreferably, the 3′-end portion sequence is from 10 to 20 T nucleotidesand can be up to 30 T nucleotides in length. Most preferably, the 3′-endportion sequence is about 15 T nucleotides in length. This primer isnamed dT15 annealing control primer (dT15-ACP). In a preferredembodiment, the first primer has a general formula of5′-dX15-30-dY2-10-dT10-20-3′, wherein dX represents adeoxyribonucleotide and comprises a pre-selected arbitrary nucleotidesequence not substantially complementary to the first and secondpopulations of mRNAs; dY represents the regulator portion comprising2-10 universal bases or non-discriminatory base analogs; and dTrepresents a contiguous deoxythymidine capable of annealing to the firstsite in the first and second populations of mRNAs.

In one embodiment, the 3′-end portion of the first ACP used in step (b)may contain at least one additional nucleotide at the 3′-end that canhybridize to an mRNA sequence which is immediately upstream of the polyAtail. The additional nucleotides at the 3′ end of the first ACP may beup to 3 in length. For example, dT may further comprise 3′-V at its3′-end; in which V is one selected from the group consisting ofdeoxyadenosine, deoxycytidine and deoxyguanosine. In addition, dT mayfurther comprise 3′-NV at its 3′-end; in which V is one selected fromthe group consisting of deoxyadenosine, deoxycytidine and deoxyguanosineand N is one selected from the group consisting of deoxyadenosine,deoxythymidine, deoxycytidine and deoxyguanosine. Most preferably, the3′-end portion sequence of the first ACP used in step (b) contains dT15only.

In a preferred embodiment, the first entire ACP is about 40-45nucleotides in length and comprises dT15 at the 3′-end portion, dX20-25at the 5′-end portion and dY5 between the 3′- and 5′-end portions. Thefirst entire ACP can be up to 100 nucleotides in length. The firstprimer is exemplified by SEQ ID NOs: 30, 39, 57 and 61-63.

The first ACP described herein is hybridized to the poly A tail of themRNA, which is present on all mRNAs, except for a small minority ofmRNA. The use of the first ACP used in this invention results in onlyone reaction and produces only one cDNA population, in contrast to atleast 3 to 64 separate cDNA populations generated by the conventionalanchor primers of Differential Display technique. This greatly increasesthe efficiency of the method by generating a substantially standard poolof single-stranded cDNA from each experimental mRNA population.

In the step (d), the standard pools of cDNAs synthesized by the firstACP should be purified and then quantitated by techniques well known tothose of ordinary skill in the art such as spectrophotometry. This stepis necessary to precisely control their inputs into the amplificationstep and then compare the final amplified poducts between two or moresamples. Preferably, the amount of cDNA produced at this point in themethod is measured. It is more preferred that this determination is madeusing ultraviolet spectroscopy, although any standard procedure knownfor quantifying cDNA known to those of ordinary skill in the art isacceptable for use for this purpose. When using the UV spectroscopyprocedure, an absorbance of about 260 nm of UV light advantageously isused. By the measurement of cDNA quantity at this step, therefore, thecDNA quantity can be standardized between or among samples in thefollowing amplification reaction.

After synthesis of the first cDNA strands using the first ACP, thesecond cDNA strands are synthesized using the second ACP primer underlow stringent conditions, by at least one cycle comprising denaturing,annealing and primer extension, wherein the resultant first cDNA strandsare used as templates.

The second ACP basically follows the rule of formula (1) and its 3′-endportion comprises a short arbitrary sequence, which preferably has lowerTm than that of the 5′-end portion. This primer is named an arbitraryannealing control primer (AR-ACP). In a preferred embodiment, the 3′-endportion of the second ACP can have from 8 to 15 nucleotides in length.Most preferably, the 3′-end portion of the second ACP contains about 10nucleotides in length.

According to a preferred embodiment, the second ACP has the generalformula of 5′-dX15-30-dY2-10-dZ8-15-3′, wherein dX represents adeoxyribonucleotide and comprises a pre-selected arbitrary nucleotidesequence not substantially complementary to the first and secondpopulations of the first cDNA strands; dY represents the regulatorportion comprising 2-10 universal bases or non-discriminatory baseanalogs; dZ represents a hybridizing arbitrary nucleotide sequencecapable of annealing to the second site in the first and secondpopulations of DNA strands. More preferably, the entire second ACP isabout 40-45 nucleotides in length comprising dZ10 at the 3′-end portion,dX20-25 at the 5′-end portion and dY5 between the 3′- and 5′-endportions. The second entire ACP can be up to 100 nucleotides in length.The second primer is exemplified by SEQ ID NOs: 1-9, 13-18 and 20-23.

The second ACP described herein is different from a so-called longarbitrary primer, as used in the known modified Differential Displaytechnique. For example, the conventional long arbitrary primers asdescribed by Villeponteau et al. (1996) and Diachenko et al. (1996),having at least 21 or 25 nucleotides in length, comprise of onlyarbitrary nucleotides in the entire sequences. These conventional longarbitrary primers will hybridize in a non-predictable way under the lowannealing temperature (about 40° C.) which is required to achievearbitrary priming in the early PCR cycle, such that it is impossible todesign a representative set of primers rationally. Furthermore, many ofthe bands represent the same mRNA due to the “Stickiness” of longprimers when used under such a low stringency.

The advantages of the present method for detecting differentiallyexpressed genes are predominantly ascribed to the use of the second ACP.Since the second ACP is designed to limit the annealing of the secondACP to its 3′-end portion sequence, not to its 5′-end portion sequence,in association with annealing temperature, the resultant annealing willcome out in a predictable way, such that it is possible to design arepresentative set of primers rationally. In addition, the use of thesecond ACP allows avoiding false positive problems caused by the“Stickiness” of the conventional long primers under low stringentconditions as used in the previous Differential Display technique.

The annealing temperature used for the synthesis of second DNA strandsunder low stringency conditions used in step (e) is preferably aboutbetween 40° C. to 65° C., more preferably, about between 45° C. and 55°C. and the most preferably, about 50° C. However, unlike DifferentialDisplay, which uses annealing temperatures between 35° C. and 45° C.,the annealing temperature of low stringency conditions used in thesubject application is relatively higher than those used in the knownclassical or enhanced Differential Display techniques with arbitraryprimers.

Another unique and significant features of the subject application fordetecting differentially expressed genes is to amplify only theinitially synthesized second DNA strands by the subsequentamplification, wherein the 3′- and 5′-ends of the second DNA strandswhich have been initially synthesized using the second ACP comprise thecomplementary sequence of the first ACP and the sequence of the secondACP, respectively and thus, the entire sequences of the first and secondACPs, or only their 5′-end portion sequences of the first and secondACPs, are used as 3′ and 5′ primer sequences for the amplification ofthe second DNA strands.

Since the ACP in the subject application leads to the amplification ofspecific products, it can be possible to fundamentally eliminate thecause of major bottleneck problems, such as false products and poorreproducibility, which result from non-specific annealing of theconventional arbitrary and dT primers to first and second DNA strands aswell as to amplified products during PCR in the known DifferentialDisplay methods.

In a preferred embodiment, the synthesis of second DNA strands in step(e) is carried out by at least 1 cycle of amplification under lowstringent conditions to achieve arbitrary priming, and through thesubsequent cycles, the amplification is processed more effectively forthe amplification of the resultant second DNA strands under highstringent conditions used in step (f). Most preferably, the synthesis ofsecond DNA strands in step (e) is carried out by one cycle ofamplification under low stringent conditions.

In a preferred embodiment, the amplification of the resultant second DNAstrands synthesized by the step (e) is carried out under high stringentconditions using the complete sequences of the first and second ACPsused in steps (b) and (e), respectively, as primer sequences, whereinthe 3′- and 5′-ends of the resultant second DNA strands provide perfectpairing sites to the first and second ACPs. However, it is interestingthat the first and second ACPs are not involved in any other annealingto the template nucleic acid, except the annealing and extension of the3′- and 5′-ends of the second DNA strands as a reaction unit at such ahigh stringent condition because their 3′-end portions requirerelatively low annealing temperature and the high stringent conditionsdo not allow them to anneal to any site of the template, except the 3′-and 5′-ends of the second DNA strands. Consequently, owing to thisfunction of ACP, which is capable of selectively annealing to thetemplate in associated with annealing temperature, the amplifiedproducts can be free from the problems of the high false positive rate,poor reproducibility and possible under-representation of minor mRNAfractions in the analysis which are the main problems of the knownDifferential Display. In this view, there is a significant differencebetween this subject method and the conventional Differential Displaymethods despite the fact that they are in common to use the same primersfor high stringent conditions as well as for low stringent conditions.

In a preferred embodiment, the annealing temperature of theamplification for high stringent conditions used in step (f) ispreferably about between 55° C. and 72° C. Most preferably, theannealing temperature used for the high stringent conditions is about65-68° C.

In a preferred embodiment, the amplification under high stringentconditions used step (f) is carried out by at least 10 cycles and up to50 cycles to amplify the resultant second DNA strands synthesized bystep (e) during PCR. Most preferably, the PCR amplification is carriedout by 40-45 cycles.

The second-strand cDNA is preferably synthesized by PCR, morepreferably, hot start PCR method in which the procedure is to set up thecomplete reactions without the DNA polymerase and incubate the tubes inthe thermal cycler to complete the initial denaturation step at >90° C.Then, while holding the tubes at a temperature above 70° C., theappropriate amount of DNA polymerase can be pipetted into the reaction.In a preferred embodiment, the addition of the primers for thesecond-stage amplification into the reaction mixture after the completereaction of the second-strand cDNA synthesis is also carried out underdenaturation temperature such as >90° C. Then, while holding the tubesat a temperature about 90° C., the appropriate amount of the primers forthe second-stage amplification can be pipetted into the reaction.

An example of the second DNA strand synthesis and the subsequentamplification of the resultant second DNA strands in a single tube usingthe pre-selected arbitrary sequence of the 5′-end portions of the firstand second ACPs is conducted under the following conditions: the secondDNA strands are synthesized under low stringent conditions by one cycleof the first-stage amplification comprising annealing, extending anddenaturing reaction; the reaction mixture containing the first-strandcDNA, PCR reaction buffer (e.g., available from Roche), dNTP, and thesecond ACP is pre-heated at about 94° C., while holding the tubecontaining the reaction mixture at about 94° C., Taq polymerase (e.g.,available from Roche) is added into the reaction mixture; the PCRreactions are as follows: one cycle of 94° C. for 1 min, 50° C. for 3min, and 72° C. for 1 min; followed by denaturing the amplificationproduct at 94° C.; after the complete reaction of the second DNA strandsynthesis in step (e), 5′ pre-selected arbitrary primer and 3′preselected arbitrary primer are added to the reaction mixture and thenthe second stage amplification is conducted as follows: 40 cycles of 94°C. for 40 sec, 68° C. for 40 sec, and 72° C. for 40 sec; followed by a 5min final extension at 72° C.

An alternative example of the second DNA strand synthesis and thesubsequent amplification of the resultant second DNA strands in a singletube using the complete sequences of the first and second ACPs used insteps (b) and (e), respectively, instead of the pre-selected arbitrarysequences of the 5′-end portions of the first and second ACPs, isconducted under the following conditions: the second DNA strands aresynthesized under low stringent conditions by one cycle of thefirst-stage amplification comprising annealing, extending and denaturingreaction; the reaction mixture containing the first-strand cDNA, PCRreaction buffer (e.g., available from Roche), dNTP, the first ACP(dT15-ACP), and the second ACP (AR-ACP) is pre-heated at about 94° C.,while holding the tube containing the reaction mixture at about 94° C.,Taq polymerase (e.g., available from Roche) is added into the reactionmixture; the PCR reactions are as follows: one cycle of 94° C. for 1min, 50° C. for 3 min, and 72° C. for 1 min; followed by thesecond-stage PCR amplification comprising annealing, extending anddenaturing reaction; the PCR reactions are as follows: 40 cycles of 94°C. for 40 sec, 65° C. for 40 sec, and 72° C. for 40 sec; followed by a 5min final extension at 72° C.

It should be noted that a proper concentration of arbitrary ACP (thesecond ACP) is used to synthesize the second-strand cDNAs by one cycleof the first-stage amplification. If the amount of the second ACP usedin the step (e) is too low, the resultant amplified products are notreproducible. In contrast, the excess amount of the second ACP used inthe step (e) generates backgrounds such as DNA smear during PCR. In apreferred embodiment, the concentration of the second ACP used in thestep (e) is about between 0.1 μM and 1.0 μM. Most preferably, theconcentration of the second ACP as well as the first ACP is about 0.2μM. In a preferred embodiment, the concentration of the primers used inthe step (f) is about between 0.1 μM and 1 μM, most preferably, about0.4 μM.

Another significant feature of the subject application to theidentification of differences in gene expression is the use of highannealing temperature in a method. High annealing temperature used instep (f) increases the specificity of primer annealing during PCR, whichresults in eliminating false positive products completely and increasingreproducibility. Freedom from false positives which is one majorbottleneck remaining for the previous Differential Display technique isespecially important in the screening step for the verification of thecDNA fragments identified by Differential Display.

The step of comparing the presence or level of amplification productsobtained from step (f) may be performed in accordance with variousmethods known in the art. In a preferred embodiment, each of the firstand second populations of amplification products of step (f) areresolved by electrophoresis to identify differentially expressed mRNAs.More preferably, the resultant PCR cDNA fragments are detected on anethidium bromide-stained agarose gel. Another prominent feature of thissubject application is the use of ethidium bromide-stained agarose gelto identify differentially expressed mRNAs. In general, the conventionalDifferential Display methods use radioactive detection techniques usingdenaturing polyacrylamide gels. However, according to the presentmethod, the significant amount of the amplified cDNA fragments obtainedthrough two stage amplifications allows to use an ethidiumbromide-stained agarose gel to detect the amplified cDNAs, which resultsin increasing the speed and avoiding the use of radioactivity.

Alternatively, the resulting cDNA fragments can be also detected on adenaturing polyacrylamide gel by autoradiography or non-radioactivedetection methods such as silver staining (Gottschlich et al., 1997;Kociok et al., 1998), the use of fluoresenscent-labelledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), and the use of biotinylated primers (Korn etal., 1992; Tagle et al., 1993; Rosok et al., 1996).

In another embodiment, it might be useful for diagnostic purposes to usean automatic system such as an automatic DNA sequencer together with anydistinct labeling of the ACPs to detect or analyze the amplifiedproducts (Bauer, et al., 1993).

Considering the features of ACP in this subject application, the presentmethod for detecting and cloning differentially expressed genes differsfundamentally from the previous Differential Display techniques asdescribed above.

In conclusion, the use of the ACP in this method makes it possible toallow the amplification of only second DNA strands and the use of thesufficient amount of starting materials as well as the highconcentration of dNTP, resulting in the following benefits: a)increasing primer annealing specificity, b) eliminating the problem offalse positives which requires the subsequent labor-intensive work toverify true positives, c) improving reliability and reproducibility, d)detecting rare mRNAs, e) generating long-distance PCR products rangingin size from 150 by to 2.0 kb, f) allowing the use of ethidiumbromide-stained agarose gel to detect products, g) increasing the speedof analysis, h) particularly, not requiring well-trained hands toconduct this method, and i) allowing the rational design of arepresentative set of primers.

In further aspect of this invention, there is provided a kit fordetecting DNA complementary to differentially expressed mRNA, whichcomprises the annealing control primer or annealing control primer setdescribed above (the first and second primer). According to oneembodiment of this invention, these kits further comprises a primer or aprimer pair having a nucleotide sequence corresponding to the 5′-endportion of the ACPs; in case that the 5′-end portion comprises universalprimer sequence, it is more preferred that the kit comprises theuniversal primers. The present kits may optionally include the reagentsrequired for performing PCR reactions such as buffers, DNA polymerase,DNA polymerase cofactors, and deoxyribonucleotide-5′-triphosphates.Optionally, the kits may also include various polynucleotide molecules,reverse transcriptase, various buffers and reagents, and antibodies thatinhibit DNA polymerase activity. The kits may also include reagentsnecessary for performing positive and negative control reactions.Optimal amounts of reagents to be used in a given reaction can bereadily determined by the skilled artisan having the benefit of thecurrent disclosure. The kits, typically, are adapted to contain inseparate packaging or compartments the constituents afore-described.

IV. Application to Rapid Amplification of cDNA Ends (RACE)

This application using the ACP of the subject invention can provide animproved method for rapidly amplifying cDNA ends, so called RACEtechnologies. To be specific, the ACP of the subject application isadapted to the RACE technologies related to either of 3′- and 5′-end,and eliminates the background problems resulting from the primers usedin the conventional RACE technologies.

In still further aspect of this invention, there is provided a methodfor rapidly amplifying a target cDNA fragment comprising a cDNA regioncorresponding to the 3′-end region of an mRNA, wherein the methodcomprises reverse transcribing said mRNA and performing an amplificationreaction using primers, characterized in that at least one primer isderived from any one of ACPs described above. Preferably, the primeraccording to the structure of ACP is one having at its 3′-end portion agene-specific hybridizing nucleotide sequence substantiallycomplementary to a site in cDNA generated from reverse transcriptionand/or one having at its 3′-end portion a hybridizing nucleotidesequence substantially complementary to poly A tails of the mRNAs.

In a specific embodiment of this invention, there is provided the methodusing two stage amplifications, which comprises:

contacting mRNAs with a first primer of any one of the ACP describedabove, in which the 3′-end portion of the primer comprises a hybridizingnucleotide sequence substantially complementary to poly A tails of themRNAs to hybridize therewith, under conditions sufficient for templatedriven enzymatic deoxyribonucleic acid synthesis to occur;

reverse transcribing the mRNAs to which the first primer hybridizes toproduce a population of first cDNA strands that are complementary to themRNAs to which the first primer hybridizes;

performing a first-stage amplification of the first cDNA strands at afirst annealing temperature comprising at least one cycle of primerannealing, primer extending and denaturing, using a second primer of anyone of the ACP described above having at its 3′-end portion agene-specific hybridizing nucleotide sequence substantiallycomplementary to a site in one of the first cDNA strands to hybridizetherewith, under conditions in which the second primer anneals to agene-specific site on one of the first cDNA strands, whereby agene-specific second cDNA strand is generated; and

performing a second-stage amplification of the gene-specific second cDNAstrand generated from step (c) at a second annealing temperature, whichis high stringent conditions, comprising at least two cycles of primerannealing, primer extending and denaturing, using the same first andsecond primers as used in steps (a) and (c), respectively, or a primerpair each comprising a pre-selected arbitrary nucleotide sequencecorresponding to each 5′-end portion of the first and second primersused in steps (a) and (c), respectively, under conditions in which eachprimer anneals to the 3′- and 5′-end sequences of a gene-specific secondcDNA strand, respectively, whereby an amplification product of agene-specific cDNA strand is generated.

Since this application using the ACP of this invention employs thepresent methods for amplification of nucleic acid sequence previouslydiscussed, the common descriptions between them are omitted in order toavoid the complexity of this specification leading to unduemultiplicity.

A schematic representation for amplifying a target cDNA fragmentcomprising 3′-end region corresponding to the 3′-end of mRNA using novelACP system, called as ACP-based 3′ RACE, is illustrated in FIG. 3.

Steps (c) and (d) of the subject application may occur in a single tubeusing the same reaction mixture except for primers, which means thatsteps (c) and (d) are separated only in time. It would be understoodthat the primers corresponding to the 5′-end portion could be added tothe reaction mixture at the time of or after second cDNA strandsynthesis. In a preferred embodiment, the primers corresponding to the5′-end portion are added to the reaction mixture right after step (2) iscompleted, followed by subsequent amplification of second cDNA strands.

As an alternative process, in step (d) the complete sequences of thefirst and second ACPs, instead of the primers corresponding to the5′-end portions of the first and second ACPs, can be used as 3′ and 5′primers for amplifying the second-strand cDNA obtained from step (c),wherein the 3′- and 5′-ends of the second-strand cDNA which areinitially synthesized using the second ACP comprise the complementarysequence of the first ACP and the sequence of the second ACP,respectively, and also serve as perfect pairing sites to the first andsecond ACPs. FIG. 3 also illustrates a schematic representation foramplifying a target cDNA fragment comprising 3′-end region correspondingto the 3′-end of mRNA by the alternative process stated above.

One of significant features of the present invention for 3′-RACE is thatthe first ACP comprising nucleotide sequence substantially complementaryto poly A tail of mRNA is used as a cDNA synthesis primer and then theresultant cDNAs are directly used as templates for subsequentamplification without any additional purification steps to remove thecDNA synthesis primer.

The annealing of the first ACP to the templates will be interruptedduring subsequent by the effect of the regulator portion on the 3′- and5′-end portions of the ACP relatively high stringent conditions asdescribed in the principle of ACP. As a result, the subject applicationto 3′-RACE simplifies the conventional RACE methods by reducing the stepof purification and also, the ACP used in the subject application doesnot involve the background problems because the annealing of the 3′-endportion is specified by the presence of the regulator portion positionedbetween the 3′- and 5′-end portions in the ACPs, whereas theconventional cDNA synthesis primers such as Oligo-dT primers for 3′-RACEgenerate backgrounds during PCR, which is non-specific products.According to a preferred embodiment, the formula of the first ACP forthe cDNA synthesis may be identical to the formula (2).

When a gene-specific primer is used as 5′ primer, the firstamplification of a target cDNA fragment containing a 3′-end sequence instep (c) is carried out in accordance with conventional PCR methods asknown in the art. The term “gene-specific” in reference sequence usedherein refers to a partial sequence of a specific gene or complementthereof that has been generally known or available to one skilled in theart. Therefore, the gene-specific primer means one comprising thegene-specific sequence.

The generated second cDNA strand is amplified by the second-stageamplification which is used in the application of the present inventionfor amplifying a target nucleic acid sequence above. Since the ACPdescribed in this invention can generate stable Tm in a primer and alsotolerate “primer search parameters” for primer design such as primerlength, annealing temperature, GC content, and PCR product length, it isuseful when the gene-specific primer sequences have low Tm or are toosensitive to such parameters to generate specific products.

In another aspect of this invention, there is provided a method forrapidly amplifying a target DNA fragment comprising a cDNA regioncorresponding to the 5′-end region of an mRNA, wherein the methodcomprises reverse transcribing the mRNA and performing an amplificationreaction using primers, characterized in that at least one primer isderived from any one of ACPs described above. Preferably, the primeraccording to the structure of ACP is one having at its 3′-end portion agene-specific hybridizing nucleotide sequence substantiallycomplementary to a site in cDNA generated from reverse transcription.

In a specific embodiment of this invention, there is provided the methodusing two stage amplifications, which comprises:

(a) contacting mRNAs with an oligonucleotide dT primer or random primeras a cDNA synthesis primer under conditions sufficient for templatedriven enzymatic deoxyribonucleic acid synthesis to occur, in which thecDNA synthesis primer comprises a hybridizing nucleotide sequencesubstantially complementary to a region of an mRNA to hybridizetherewith;

(b) reverse transcribing the mRNAs, using a reverse transcriptase, towhich the cDNA synthesis primer hybridizes to produce a population offirst cDNA strands that are complementary to the mRNAs to which the cDNAsynthesis primer hybridizes, whereby mRNA-cDNA intermediates aregenerated;

(c) permitting cytosine residues to be tailed at the 3′-ends of thefirst cDNA strands in the form of the mRNA-cDNA intermediates by theterminal transferase reaction of reverse transcriptase;

(d) contacting the cytosine tails at the 3′-ends of the first cDNAstrands generated from step (c) with an oligonucleotide which comprisesa 3′-end portion and a 5′-end portion separated by a group of universalbase or non-discriminatory base analog, wherein the 3′-end portioncomprises at least three guanine residues at its 3′-end to hybridizewith the cytosine tails at the 3′-ends of the first cDNA strands and the5′-end portion comprises a pre-selected arbitrary nucleotide sequence,under conditions in which the 3-end portion of the oligonucleotide ishybridized to the cytosine tails;

(e) extending the tailed 3′-ends of the first cDNA strands to generatean additional sequence complementary to the oligonucleotide usingreverse transcriptase, in which the oligonucleotide serves as a templatein the extension reaction, whereby full-length first cDNA strands areextended;

(f) performing a first-stage amplification of the full-length first cDNAstrands obtained from step (e) at a first annealing temperaturecomprising (i) and (ii) as follows:

(i) at least one cycle of primer annealing, primer extending anddenaturing using a first primer comprising a nucleotide sequencesubstantially complementary to the 3′-end sequences of the full-lengthfirst cDNA strands under conditions in which the first primer anneals tothe full-length first cDNA strands, under conditions in which the firstprimer anneals to the 3′-ends of the full-length first cDNA strands,whereby full-length second cDNA strands are generated;

(ii) at least one cycle of primer annealing, primer extending anddenaturing using a second primer of any one of claims 1-25 having at its3′-end portion a gene-specific hybridizing sequence substantiallycomplementary to a region on one of the full-length second cDNA strandsto hybridize therewith, under conditions in which the second primeranneals to a gene-specific site on one of the full-length second cDNAstrands, whereby a gene-specific cDNA strand is generated; and

(g) performing a second-stage amplification of the gene-specific cDNAstrand at a second annealing temperature, which is high stringentconditions, comprising at least two cycles of primer annealing, primerextending and denaturing, using the same first and second primers asused in steps (f)-(i) and (f)-(ii), respectively, or a primer pair eachcomprising a nucleotide sequence corresponding to each 5′-end portion ofthe first and second primers as used in steps (f)-(i) and (f)-(ii),respectively, under conditions in which each primer anneals to the 3′-and 5′-end sequences of a gene-specific cDNA strand, respectively,whereby an amplification product of a gene-specific cDNA strand isgenerated.

The schematic representations for amplifying a target cDNA fragmentcomprising 5′-end region corresponding to the 5′-end of mRNA using novelACP system, called as ACP-based 5′ RACE, is illustrated in FIGS. 4A(using oligonucleotide dT primer) and 4B (using random primer).

The descriptions of the oligonucleotide dT primer used in step (a) isidentical to those used in the present method for amplification of atarget nucleic acid sequence from an mRNA. Alternatively, when the sizeof a target mRNA is so large that the reverse transcriptase falls offbefore reaching the 5′ complete sequences, the random primer is used ascDNA synthesis primer.

According to a preferred embodiment, the step (c), permitting cytosineresidues to be tailed is performed in the presence of manganese ion.

Steps (f) and (g) of the subject application may occur in a single tubeusing the same reaction mixture except for primers, which means thatsteps (f) and (g) are separated only in time. It would be understoodthat the primer(s) used in each step (f) and (g) can be added to thereaction mixture at the time of or after each step. In a preferredembodiment, the primer(s) is(are) added to the reaction mixture rightafter each step is completed, followed by subsequent PCR amplificationof second cDNA strands.

When a gene-specific primer is used as 5′ primer, the amplification of atarget cDNA fragment containing a 5′-end sequence in step (f) is carriedout under high stringent conditions in accordance with conventional PCRmethods as known in the art.

In a preferred embodiment, a target cDNA fragment containing a 5′-endsequence in step (f) is amplified using a second ACP comprising agene-specific sequence at the 3′-end portion, by two stage PCRamplifications which are used in the application of the presentinvention for amplifying a target nucleic acid sequence above. Since theACP described in this invention can generate stable Tm in a primer andalso tolerate “primer search parameters” such as primer design,comprising primer length, annealing temperature, GC content, and PCRproduct length, it is particularly useful when the gene-specific primersequences have low Tm or are too sensitive to such parameters togenerate specific products. The formula of the second ACP is identicalto the formula (1) in which the 3′-end portion contains a gene-specificsequence.

The oligonucleotide for the step (d) is similar to CapFinder primer(Chenchik et al., 1998; Chenchik et al. U.S. Pat. Nos. 5,962,271 and5,962,272) in the senses both of them comprise at least three guanineresidues at its 3′-end and use them as a template switching primer forthe 3′-end extension of the first cDNA strand by reverse transcriptase,whereas they are clearly different from each other in terms of thefunction of a switch in controlling primer annealing to a templatenucleic acid in associated with annealing temperature during PCR.CapFinder primer does not comprise univeasl base residue group which isresponsible for regulating primer annealing in ACP, so that theCapFinder PCR method for 5′-RACE (Chenchik et al., 1998) can not be freefrom a high background such as DNA spear arising from contamination ofthe primers such as the CapFinder and Oligo-dT primers used in cDNAsynthesis during PCR. On the other hand, the universal base resiudegroup of the first ACP plays a key role in regulating primer annealing,so that the subject method does not provide any cause for the backgroundproblems during subsequent PCR amplification; this is a key feature ofthe ACP application to 5′-RACE.

Furthermore, when the ACP of the present invention is used in 5′-RACEtechnology, it is unnecessary to conduct the process of physicalseparation such as a solid-phase cDNA synthesis and procedures which hasbeen introduced as an alternative method to remove all contaminants usedin cDNA synthesis (Schramm et al., 2000).

In a preferred embodiment, the oligonucleotide to form a base-pair(s)with the cytosine tail for 5′-RACE which has a similar structure to ACP,wherein the oligonucleotide is represented by the following generalformula (3): 5′-dX15-30-dY2-10-dZI-10-G3-5-3′, in which dX represents adeoxyribonucleotide and comprises a preselected arbitrary sequence; dYrepresents a regulatory portion comprising 2-10 universal bases ornon-discriminatory base analogs; dZ represents a deoxyribonucleotide andcomprises a pre-selected arbitrary sequence; and G3-5 represents threeto five guanines.

Most preferably, the 3′-end portion sequence dZ is about 2-3 nucleotidesin length. Further, in one embodiment, the 5′-end portion dX can includea sequence that is recognized by a restriction endonuclease.

The G3-5 may be three to five riboguanines or deoxyguanines, or acombination of riboguanine and deoxyriboguanine. In more preferredembodiment, the G3-5 comprises two riboguanines and one deoxyriboguanine(r(G)₂-d(G)-3′), most preferably, three riboguanines.

When the gene-specific primer in step (f) is used as 3′ primer for5′-RACE, a target cDNA fragment containing a 5′-end sequence isamplified under high stringency conditions by conventional PCR methodsas known in the art.

In a preferred embodiment, a target cDNA fragment containing a 5′-endsequence is amplified using a second ACP which comprises a gene-specificsequence at the 3′-end portion, by two stage PCR amplifications which isconducted in the application for amplifying a target nucleic acidsequence in the present invention. Since the ACP described in thisinvention can provide stable Tm in a primer and also tolerate “primersearch parameters” for primer design such as primer length, annealingtemperature, GC content, and PCR product length, it is useful when thegene-specific primer sequences have low Tm or are too sensitive to suchparameters to generate specific products. The formula of the second ACPis identical to the formula (1) in which the 3′-end portion contains agene-specific sequence.

The use of ACP in RACE technology significantly simplifies and improvesthe conventional RACE technologies with regard to the amplification ofcDNA ends as described above. The vital feature of the subject method isto be free from the background problems arising from the primers used inconventional RACE methods. Consequently this method described herein canbe more effective, easier, less labor-intensive, and more reproduciblethan conventional RACE methods.

In still another aspect of this invention, there is provided a kit forrapidly amplifying a target cDNA fragment comprising 3′-end region ofmRNA, which comprises the annealing control primer or annealing controlprimer set described previously (including the first and second primer).According to one embodiment of this invention, these kits furthercomprises a primer or a primer pair having a nucleotide sequencecorresponding to the 5′-end portion of the ACPs; in case that the 5′-endportion comprises universal primer sequence, it is more preferred thatthe kit comprises the universal primers. The present kits may optionallyinclude the reagents required for performing PCR reactions such asbuffers, DNA polymerase, DNA polymerase cofactors, anddeoxyribonucleotide-5′-triphosphates. Optionally, the kits may alsoinclude various polynucleotide molecules, reverse transcriptase, variousbuffers and reagents, and antibodies that inhibit DNA polymeraseactivity. The kits may also include reagents necessary for performingpositive and negative control reactions. Optimal amounts of reagents tobe used in a given reaction can be readily determined by the skilledartisan having the benefit of the current disclosure. The kits,typically, are adapted to contain in separate packaging or compartmentsthe constituents afore-described.

In further aspect of this invention, there is provided a kit for rapidlyamplifying a target cDNA fragment comprising 5′-end region of mRNA,which comprises the annealing control primer or annealing control primerset described above (including the oligonucleotide dT primer and randomprimer for cDNA synthesis, the oligonucleotide to form a base-pair(s)with the cytosine tail, the first primer and the second primer).According to one embodiment of this invention, these kits furthercomprises a primer pair each comprising a nucleotide sequencecorresponding to each 5′-end portion of the first and second primers asused in steps (f)-(i) and (f)-(ii); in case that the 5′-end portioncomprises universal primer sequence, it is more preferred that the kitcomprises the universal primers.

V. Application to Amplifying Full-Length cDNA

In further aspect of this invention, there is provided a method foramplifying a population of full-length double-stranded cDNAscomplementary to mRNAs, wherein the method comprises reversetranscribing the mRNA and performing an amplification reaction usingprimers, characterized in that at least one primer is derived from anyone of ACP described above. Preferably, the primer having the structureof ACP is one having a hybridizing nucleotide sequence substantiallycomplementary to poly A tails of mRNAs.

In a specific embodiment of this invention, there is provided the methodcomprises:

(a) contacting the mRNAs with a first primer of any one of ACP describedabove, in which the 3′-end portion of the first primer has a hybridizingnucleotide sequence substantially complementary to poly A tails of themRNAs to hybridize therewith, under conditions sufficient for templatedriven enzymatic deoxyribonucleic acid synthesis to occur;

(b) reverse transcribing the mRNAs, using a reverse transcriptase, towhich the first primer hybridizes to produce the population of firstcDNA strands that are complementary to the mRNAs to which the primerhybridizes, whereby mRNA-cDNA intermediates are generated;

(c) permitting cytosine residues to be tailed at the 3′-ends of thefirst cDNA strands in the form of the mRNA-cDNA intermediates by theterminal transferase reaction of reverse transcriptase;

(d) contacting the cytosine tails at the 3′-ends of the first cDNAstrands generated from step (c) with an oligonucleotide which comprisesa 3′-end portion and a 5′-end portion separated by a group of universalbase or non-discriminatory base analog, wherein the 3′-end portioncomprises at least three guanine residues at its 3′-end to hybridizewith the cytosine tails at the 3′-ends of the first cDNA strands and the5′-end portion comprises a pre-selected arbitrary nucleotide sequence,under conditions in which the 3-end portion of the oligonucleotide ishybridized to the cytosine tails;

(e) extending the tailed 3′-ends of the first cDNA strands to generatean additional sequence complementary to the oligonucleotide usingreverse transcriptase, in which the oligonucleotide serves as a templatein the extension reaction, whereby full-length first cDNA strands areextended; and

(f) performing an amplification of the full-length first cDNA strandsgenerated from step (e) comprising at least two cycles of primerannealing, primer extending and denaturing, using a primer pair eachcomprising a nucleotide sequence corresponding to the same first primerand oligonucleotide as used in steps (a) and (d), respectively, or aprimer pair each comprising a nucleotide sequence corresponding to each5′-end portion of the first primer and oligonucleotide used in steps (a)and (d), respectively, under conditions in which

(g) each primer anneals to the 3′- and 5′-end sequences of thefull-length first cDNA strands, respectively, whereby amplificationproducts of full-length cDNA strands complementary to the mRNAs aregenerated.

Since this application using the ACP of this invention employs inprinciple the present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity. In addition, the ACP described above in which the 3′-endportion has a hybridizing nucleotide sequence substantiallycomplementary to poly A tails is in principle identical to the firstprimer for the present method for 3′-RACE. Furthermore, theoligonucleotide to form a base-pair(s) with the cytosine tail and theprimer pair used in the step (f) are in principle identical to those for5′-RACE of this invention discussed above.

A schematic representation for amplifying full-length cDNA molecules ofthe present invention is illustrated in FIG. 5.

The use of ACP significantly simplifies and improves the conventionaltechnologies with regard to the amplification of full-length cDNAs asdescribed above. The vital feature of the subject method is to be freefrom the background problems arising from the primers used inconventional methods. Consequently this method described herein can bemore effective, easier, less labor-intensive, and more reproducible thanconventional methods.

In still further aspect of this invention, there is provided a kit foramplifying a full-length double stranded cDNA complementary to mRNA,which comprises the annealing control primer or the annealing controlprimer set described above (including the oligonucleotide dT primer, theoligonucleotide to form a base-pair(s) with the cytosine tail, theprimer(s) used in the step (f). According to one embodiment of thisinvention, these kits further comprises a primer pair each comprising anucleotide sequence corresponding to each 5′-end portion of the primerand oligonucleotide used in steps (a) and (d), respectively; in casethat the 5′-end portion comprises universal primer sequence, it is morepreferred that the kit comprises the universal primers. The present kitsmay optionally include the reagents required for performing PCRreactions such as buffers, DNA polymerase, DNA polymerase cofactors, anddeoxyribonucleotide-5′-triphosphates. Optionally, the kits may alsoinclude various polynucleotide molecules, reverse transcriptase, variousbuffers and reagents, and antibodies that inhibit DNA polymeraseactivity. The kits may also include reagents necessary for performingpositive and negative control reactions. Optimal amounts of reagents tobe used in a given reaction can be readily determined by the skilledartisan having the benefit of the current disclosure. The kits,typically, are adapted to contain in separate packaging or compartmentsthe constituents afore-described.

VI. Application to Amplifying 5′-Enriched cDNA

In another aspect of this invention, there is provided a method foramplifying a population of 5′-enriched double-stranded cDNAs comprisingcDNA regions corresponding to the 5′-end regions of mRNAs, wherein themethod comprises reverse transcribing the mRNA and performing anamplification reaction using primers, characterized in that at least oneprimer is derived from any one of ACP described above. Preferably, theprimer having the structure of ACP used for cDNA synthesis is one havingat its 3′-end portion at least six random nucleotide sequences.

In a specific embodiment of this invention, there is provided the methodcomprises:

(a) contacting the mRNAs with a first primer of any one of ACP describedabove under conditions sufficient for template driven enzymaticdeoxyribonucleic acid synthesis to occur, wherein the 3′-end portion ofthe first primer has at least six random nucleotide sequences;

(b) performing the steps (b)-(e) of the method for amplifying apopulation of full-length double-stranded cDNAs, whereby 5′-enrichedfirst cDNA strands are extended;

(c) performing an amplification of the 5′-enriched first cDNA strandsgenerated from step (b) comprising at least two cycles of primerannealing, primer extending and denaturing, using a primer pair eachcomprising a nucleotide sequence corresponding to each 5′-end portion ofthe primer and oligonucleotide used in steps (a) and (b), respectively,under conditions in which each primer anneals to the 3′- and 5′-endsequences of the 5′-enriched first cDNA strands, respectively, wherebyamplification products of 5′-enriched cDNA strands are generated.

Since this application using the ACP of this invention employs inprinciple the present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity. In addition, the oligonucleotide to form a base-pair(s)with the cytosine tail and the primer pair used in the step (c) are inprinciple identical to those for 5′-RACE of this invention discussedabove.

A schematic representation for the method for amplifying 5′-enricheddouble-stranded cDNAs complementary to mRNAs is illustrated in FIG. 6.

“5′ enriched cDNAs” refers to a significant portion of the cDNAconstituents which contain the nucleotide sequence information of the5′-end of the mRNAs from which the cDNAs are derived.

The formula of the first primer is identical to the formula (1) in whichthe 3′-end portion comprises a random nucleotide sequence. In apreferred embodiment, the 3′-end portion of the first primer used instep (a) contains at least six random deoxyribonucleotides. In apreferred embodiment, the 5′-end portion of the first primer used instep (a) can includes a sequence that is recognized by a restrictionendonuclease.

The conventional methods require more steps to amplify 5′ enriched cDNAmolecules complementary to the mRNA molecules than the subject methodbecause the conventional methods use the conventional primers which donot have the function of controlling primer annealing. In contrast, thissubject method is considerably a simple and effective approach due tothe function of regulating primer annealing generated by the effect of auniversal base residue group in ACP.

In still another aspect of this invention, there is provided a kit foramplifying 5′-enriched double-stranded cDNAs complementary to mRNAs,which comprises the annealing control primer or the annealing controlprimer set described above (the first primer, the oligonucleotide toform a base-pair(s) with the cytosine tail). According to one embodimentof this invention, these kits further comprises a primer pair eachcomprising a nucleotide sequence corresponding to each 5′-end portion ofthe primer and oligonucleotide used in steps (a) and (b), respectively;in case that the 5′-end portion comprises universal primer sequence, itis more preferred that the kit comprises the universal primers. Thepresent kits may optionally include the reagents required for performingPCR reactions such as buffers, DNA polymerase, DNA polymerase cofactors,and deoxyribonucleotide-5′-triphosphates. Optionally, the kits may alsoinclude various polynucleotide molecules, reverse transcriptase, variousbuffers and reagents, and antibodies that inhibit DNA polymeraseactivity. The kits may also include reagents necessary for performingpositive and negative control reactions. Optimal amounts of reagents tobe used in a given reaction can be readily determined by the skilledartisan having the benefit of the current disclosure. The kits,typically, are adapted to contain in separate packaging or compartmentsthe constituents afore-described.

VII. Application to DNA or RNA Fingerprinting

This application using ACP of the subject invention can provide animproved method for detecting polymorphisms in genomic DNA (DNAfingerprinting) or for detecting differential gene expression in mRNA(RNA fingerprinting).

In further aspect of this invention, there is provided a method forproducing a DNA fingerprint of gDNA, wherein the method comprisesperforming an amplification reaction using primers, characterized inthat at least one primer is derived from any one of ACPs describedabove. Preferably, the primer having the structure of ACP is one havingat its 3′-end portion an arbitrary nucleotide sequence substantiallycomplementary to sites on the gDNA.

In a specific embodiment of this invention, there is provided the methodusing two stage amplifications, which comprises:

(a) performing a first-stage amplification of the DNA fingerprint, whichis a set of discrete DNA segments characteristic of genome, from thegDNA at a first annealing temperature comprising at least two cycles ofprimer annealing, primer extending and denaturing, using the primer orthe primer pair of any one of ACPs described above, wherein each primerhas at its 3′-end portion an arbitrary nucleotide sequence substantiallycomplementary to sites on the gDNA to hybridize therewith, underconditions in which the primer or the primer pair anneals to the gDNA,whereby the set of discrete DNA segments characterized as a DNAfingerprint is produced; and

(b) performing a second-stage amplification of the set of discrete DNAsegments generated from step (a) at a second annealing temperature,which is high stringent conditions, comprising at least one cycle ofprimer annealing, primer extending and denaturing, using the same primeror primer pair as used in step (a) or a primer or a primer pair eachcomprising a nucleotide sequence corresponding to each 5′-end portion ofthe primer or primer pair used in step (a), under conditions in whichthe primer or each of the primer pair anneals to the 3′- and 5′-endsequences of the set of discrete DNA segments generated from step (a),respectively, whereby the set of discrete DNA segments is re-amplified.

In still further aspect of this invention, there is provided a methodfor producing a RNA fingerprint of an mRNA sample, wherein the methodcomprises reverse transcribing and performing an amplification reactionusing primers, characterized in that at least one primer is derived fromany one of ACPs. Preferably, the primer according to the structure ofACP is one having at its 3′-end portion an arbitrary nucleotide sequencesubstantially complementary to sites on cDNA strands generated fromreverse transcription and/or one having at its 3′-end portion ahybridizing nucleotide sequence substantially complementary to poly Atails of the mRNAs.

In a specific embodiment of this invention, there is provided the methodusing two stage amplifications, which comprises:

(a) contacting the mRNA sample with a first primer of any one of ACPsdescribed above, in which the first primer has a hybridizing nucleotidesequence substantially complementary to poly A tails of the mRNA sampleto hybridize therewith, under conditions sufficient for template drivenenzymatic deoxyribonucleic acid synthesis to occur;

(b) reverse transcribing the mRNA sample to which the first primerhybridizes to produce a population of first cDNA strands that arecomplementary to the mRNA sample to which the first primer hybridizes;

(c) performing a first-stage amplification of the population of firstcDNA strands generated from step (b) at a first annealing temperaturecomprising at least one cycle of primer annealing, primer extending anddenaturing, using a second primer or primer pair of any one of ACPsdescribed above, wherein each primer has at its 3′-end portion anarbitrary nucleotide sequence substantially complementary to sites onthe first cDNA strands to hybridize therewith, under conditions in whichthe primer or primer pair anneals to the mRNA sample, whereby a set ofdiscrete cDNA segments characterize as a RNA fingerprint is produced;and

(d) performing a second stage amplification of the set of discrete cDNAsegments generated from step (c) at a second annealing temperature whichis high stringent conditions, comprising at least one cycle of primerannealing, primer extending and denaturing, using the same primer orprimer pair as used in step (c) or a primer or primer pair eachcomprising a nucleotide sequence corresponding to each 5′-end portion ofthe primer or primer pair used in step (c), under conditions in whichthe primer or each of the primer pair anneals to the 3′- and 5′-endsequences of the set of discrete cDNA segments generated from step (c),respectively, whereby the set of discrete cDNA segments is re-amplified.

Since this application using the ACP of this invention employs inprinciple the present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity. In addition, the RNA fingerprinting in principle followsthe present method for detecting DNA complementary to differentiallyexpressed mRNA.

The term “genomic DNA” as used herein refers to a population of DNA thatcomprises the complete genetic component of a species. Thus genomic DNAcomprises the complete set of genes present in a pre-selected species.The complete set of genes in a species is also referred to as genome.The term DNA or RNA “fingerprinting” as used herein refers to a set ofdiscrete DNA amplification products characteristic of a genome or a setof discrete cDNA segments characteristic of a sample of mRNA,respectively.

In the previous arbitrarily primed PCR fingerprints, called AP-PCR,short or long arbitrary primers have been used under non-stringentconditions for early 2-5 cycles of PCR amplification because a lowannealing temperature is required to achieve arbitrary priming, suchthat a significant portion of isolated fragments is not stillreproducible although effective amplification proceeds in the followingcycles under high stringent condition.

In contrast to AP-PCR, the ACP-based PCR for fingerprinting increasesthe specificity of primer annealing during PCR due to the function of auniversal base residue group positioned between the 3′- and 5′-endportions of ACP, wherein the universal base residue group restricts theannealing site to the 3′-end portion of the ACP and also allows this3′-end portion to anneal at a relatively high annealing temperature.Thus, the ACP-based PCR for fingerprinting completely eliminates falsepositive products and significantly increases reproducibility.

In a preferred embodiment, the ACP contains an arbitrary sequence at the3′-end portion with at least 6 nucleotides in length. More preferably,the 3′-end portion contains 8-15 nucleotides in length, most preferably,about 10 nucleotides in length.

A single ACP or a pair of ACPs can be used for detecting polymorphismsin DNA fingerprinting. Preferably, a pair of ACPs is used for DNAfingerprinting because a pair of ACPs produces more products than asingle arbitrary ACP does.

An example of the DNA fingerprinting using ACP is conducted by twostages of PCR amplifications under the following conditions:amplification reactions are performed under low stringent conditions bytwo cycles of the first-stage PCR comprising annealing, extending anddenaturing reaction; the reaction mixture containing genomic DNA, PCRreaction buffer, MgCl2, dNTPs (dATP, dCTP, dGTP and dTTP), a pair ofACPs is pre-heated, Taq polymerise is added into the reaction mixture;the PCR reactions are performed, followed by denaturing theamplification product; after the complete reaction of the first-stagePCR, the pre-selected arbitrary primer JYC4 corresponding to the 5′-endportion of the ACPs are added to the reaction mixture and then thesecond stage PCR amplification is conducted.

It should be noted that a proper concentration of ACP is used to produceDNA fingerprinting. If the amount of the ACP is too low, the resultantamplified PCR products are not reproducible. In contrast, the excessamount of the ACP generates backgrounds such as DNA smear during PCR. Ina preferred embodiment, the concentration of the ACP is about between0.1 μM and 2 μM. Most preferably, the concentration of the ACP is about1.4 μM.

In a preferred embodiment, the concentration of the primer correspondingto the 5′-end portion of the ACPs is about between 0.1 μM and 2 μM, mostpreferably, about 0.8 μM.

The genomic DNA and mRNA samples may be obtained from a wide variety ofbiomaterials and conditions. For example, they may be obtained fromplants, animal (human) and microbes and from different individualorganisms.

The amplified products can be analyzed by gel electrophoresis. In oneembodiment, the resulting PCR products can be also detected on adenaturing polyacrylamide gel by autoradiography or non-radioactivedetection methods such as silver staining (Gottschlich et al., 1997;Kociok et al., 1998), the use of fluoresenscent-labelledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), and the use of biotinylated primers (Korn etal., 1992; Tagle et al., 1993; Rosok et al., 1996).

In still further aspect of this invention, there is provided a kit forproducing a DNA fingerprint by use of gDNA or mRNA, which comprises theannealing control primer or annealing control primer set describedabove. The descriptions of the kits for the amplification of nucleicacid sequence and for detecting DNA complementary to differentiallyexpressed mRNA of this invention can be applied to the present kit.

VIII. Application to Identification of Conserved Homology Segments inMultigene Families

This application using ACP of the subject invention can also provide animproved method for the identification of conserved homology segments inmultigene families.

In another aspect of this invention, there is provided a method foridentifying conserved homology segments in a multigene family from anmRNA sample, wherein the method comprises reverse transcribing andperforming an amplification reaction using primers, characterized inthat at least one primer is derived from any one of ACPs describedabove. Preferably, the primer having the structure of ACP is one havingat its 3′-end portion a hybridizing sequence substantially complementaryto a consensus sequence or a degenerate sequence encoding amino acidsequence of a conserved homology segment on cDNA strands generated fromreverse transcription and/or one having at its 3′-end portion ahybridizing nucleotide sequence substantially complementary to poly Atails of the mRNAs.

In a specific embodiment of this invention, there is provided the methodusing two stage amplifications, which comprises:

(a) contacting the mRNA sample with a first primer of any one of claims1-29, in which the first primer has a hybridizing nucleotide sequencesubstantially complementary to poly A tails of the mRNA sample tohybridize therewith, under conditions sufficient for template drivenenzymatic deoxyribonucleic acid synthesis to occur;

(b) reverse transcribing the mRNA sample to which the first primerhybridizes to produce a population of first cDNA strands that arecomplementary to the mRNA sample to which the first primer hybridizes;

(c) performing a first-stage amplification of the population of firstcDNA strands generated from step (b) at a first annealing temperaturecomprising at least one cycle of primer annealing, primer extending anddenaturing, using a second primer of any one of claims 1-25 having atits 3′ end portion a hybridizing sequence substantially complementary toa consensus sequence or a degenerate sequence encoding amino acidsequence of a conserved homology segment on the first cDNA strands tohybridize therewith, under conditions in which the second primer annealsto the consensus sequence or degenerate sequence of first cDNA strands,whereby 3′-end cDNA segments having the consensus sequence or degeneratesequence are generated; and

(d) performing a second stage amplification of the 3′-end cDNA segmentsgenerated from step (c) at a second annealing temperature which is highstringent conditions, comprising at least two cycles of primerannealing, primer extending and denaturing, using the same first andsecond primers as used in steps (a) and (c) or a primer pair eachcomprising a nucleotide sequence corresponding to each 5′-end portion ofthe first and second primers used in steps (a) and (c), respectively,under conditions in which each primer anneals to the 3′- and 5′-endsequences of the 3′-end cDNA segments, respectively, whereby the 3′-endconserved homology cDNA segments are amplified.

This specific embodiment follows in principle, the present method for 3′RACE as discussed previously except for the second primer used.

In another specific embodiment of this invention, there is provided themethod using two stage amplifications, which comprises:

(a) performing steps of (a)-(e) of the method for amplifying apopulation of full-length double-stranded cDNA, whereby full-length cDNAstrands are generated;

(b) performing a first-stage amplification of the full-length first cDNAstrands obtained from step (a) at a first annealing temperature, whichcomprises the steps of:

-   -   (i) at least one cycle of primer annealing, primer extending and        denaturing using a first primer comprising a nucleotide sequence        substantially complementary to the 3′-end sequences of the        full-length first cDNA strands under conditions in which the        first primer anneals to the full-length first cDNA strands,        under conditions in which the first primer anneals to the        3′-ends of the full-length first cDNA strands, whereby        full-length second cDNA strands are generated; and    -   (ii) at least one cycle of primer annealing, primer extending        and denaturing using a second primer of any one of claims 1-25        having at its 3′ end portion a hybridizing sequence        substantially complementary to a consensus sequence or a        degenerate sequence encoding amino acid sequence of a conserved        homology segment on the full-length second cDNA strands to        hybridize therewith, under conditions in which the second primer        anneals to the consensus sequence or degenerate sequence of        full-length second cDNA strands, whereby 5′-end cDNA segments        having the consensus sequence or degenerate sequence are        generated; and

(c) performing a second stage amplification of the 5′-end cDNA segmentsgenerated from step (b) at a second annealing temperature which is highstringent conditions, comprising at least two cycles of primerannealing, primer extending and denaturing, using the same first andsecond primers as used in steps (b)-(i) and (b)-(ii), respectively, or aprimer pair each comprising a nucleotide sequence corresponding to each5′-end portion of the first and second primers used in steps (b)-(i) and(b)-(ii), respectively, under conditions in which each primer anneals tothe 3′- and 5′-end sequences of the 5′-end cDNA segments, respectively,whereby the 5′-end conserved homology cDNA segments are amplified.

This specific embodiment follows in principle, the present method for 5′RACE as discussed previously except for the second primer used.

In further aspect of this invention, there is provided a method foridentifying conserved homology segments in a multigene family from gDNA,wherein the method comprises performing an amplification reaction usingprimers, characterized in that at least one primer is derived from anyone of ACPs described above. Preferably, the primer having the structureof ACP is one having at its 3′-end portion a hybridizing sequencesubstantially complementary to a consensus sequence or a degeneratesequence encoding amino acid sequence of a conserved homology segment onthe gDNA.

In a specific embodiment of this invention, there is provided the methodusing two stage amplifications, which comprises:

(a) performing a first-stage amplification of the conserved homologysegments from the gDNA at a first annealing temperature comprising atleast two cycles of primer annealing, primer extending and denaturing,using the primer or the primer pair of any one of ACPs described above,wherein each primer has at its 3′ end portion a hybridizing sequencesubstantially complementary to a consensus sequence or a degeneratesequence encoding amino acid sequence of a conserved homology segment onthe gDNA to hybridize therewith, under conditions in which the primer orthe primer pair anneals to the consensus sequence or degenerate sequenceof gDNA, whereby genomic DNA segments having the consensus sequence ordegenerate sequence are generated; and

(b) performing a second-stage amplification of the genomic DNA segmentsgenerated from step (a) at a second annealing temperature, which is highstringent conditions, comprising at least one cycle of primer annealing,primer extending and denaturing, using the same primer or primer pair asused in step (a) or a primer or a primer pair each comprising anucleotide sequence corresponding to each 5′-end portion of the primeror primer pair used in step (a), under conditions in which the primer oreach of the primer pair anneals to the 3′- and 5′-end sequences of thegenomic DNA segments generated from step (a), respectively, whereby theconserved homology genomic segments are amplified.

The present method follows in principle, the present method foramplifying a target nucleic acid sequence from a DNA as discussedpreviously except for the primer used.

Since this application using the ACP of this invention employs inprinciple the present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity. In addition, where an mRNA is used as starting material,the present methods for 3′ or 5′ RACE are in principle applied to thepresent methods for the identification of conserved homology segments inmultigene families.

The formula of ACP for the identification of conserved homology segmentsin multigene families is identical to the formula (1) in which the3′-end portion of ACP has a hybridizing sequence substantiallycomplementary to a consensus sequence in a gene family or a degeneratesequence encoding amino acid sequence of a conserved homology.

There are two principle approaches to the design of degenerate primer:

(a) using peptide sequence data obtained from a purified protein; and

(b) using consensus protein sequence data from alignments of genefamilies.

If orthologs of the gene of interest have been cloned from otherorganisms, or if the gene is a member of a gene family, it will bepossible to generate protein sequence alignments.

These may reveal appropriate regions for the design of degenerateprimers, for example, from consensus sequence of highly conservedregions. Amplifications using degenerate primers can sometimes beproblematic and may require optimization. The first parameter isannealing temperature. It is important to keep the annealing temperatureas high as possible to avoid extensive nonspecific amplification and agood rule of thumb is to use 55° C. as a starting temperature. Ingeneral, it is difficult to keep this rule because degenerate primersshould be designed based on amino acid sequences as a precondition.However, the ACP of the present invention does not have to satisfy thisrequirement because it allows a high annealing temperature such as 65°C. at the second stage of PCR amplification regardless of primer design.

According to a preferred embodiment, the second primer is a pool ofprimers each comprising a degenerate sequence selected from a pluralityof the nucleotides coding for amino acid sequence of the consensussequence.

The term “conserved region” and more specifically “conserved region of agene in a multigene family” as used herein refers to a segment ofnucleotide sequence of a gene or amino acid sequence of a protein thatis significantly similar between members of gene families. The degree ofsimilarity can vary. In some cases the conserved regions will beidentical between family members. In some cases the nucleotide sequencemay vary significantly but still encode for amino acid segments that areconserved between family members. The term “consensus sequence” as usedherein refers to the bases most often found at any given position whencomparing a large number of similar nucleotide sequences.

Alternatively, the present methods for the identification of conservedhomology segments can be also combined with that for detectingdifferentially expressed mRNAs.

In still further aspect of this invention, there is provided a kit foridentifying a conserved homology segment in a multigene family by use ofmRNA or gDNA, which comprises the annealing control primer or annealingcontrol primer set described above. The descriptions of the kits for theamplification of nucleic acid sequence, 3′ RACE and 5′ RACE of thisinvention can be applied to the present kit.

IX. Application to Identification of a Nucleotide Variation

This application using ACP system of the subject invention can alsoprovide an improved method for identifying a nucleotide variation in atarget nucleic acid.

In another aspect of this invention, there is provided a method foridentifying a nucleotide variation in a target nucleic acid, wherein themethod comprises performing an amplification reaction using primers,characterized in that at least one primer is derived from any one ofACPs described above. Preferably, the primer having the structure of ACPis (a) a first primer one having at its 3′-end portion a hybridizingsequence substantially complementary to a pre-selected sequence at afirst site of target nucleic acid, wherein each of the first primer andthe first site comprises an interrogation position corresponding to thenucleotide variation, and/or (b) a second primer having a hybridizingsequence substantially complementary to a pre-selected sequence at asecond site of target nucleic acid.

In a specific embodiment of this invention, there is provided the methodusing two stage amplifications, which comprises:

(a) performing a first-stage amplification to produce a first DNA strandcomplementary to the target nucleic acid including the nucleotidevariation at a first annealing temperature comprising at least one cycleof primer annealing, primer extending and denaturing, using a firstprimer of any one of ACPs described above having at its 3′-end portion ahybridizing sequence substantially complementary to a pre-selectedsequence at a first site of the target nucleic acid to hybridizetherewith, wherein each of the first primer and the first site comprisesan interrogation position corresponding to the nucleotide variation,whereby the first DNA strand complementary to the target nucleic acidincluding the nucleotide variation is generated when the interrogationposition is occupied by the complementary nucleotide of the first primerto its corresponding nucleotide of the first site; and

(b) performing a second-stage amplification of the first DNA strandgenerated from step (a) at a second annealing temperature, which is highstringent conditions, comprising the steps:

-   -   (i) at least one cycle of primer annealing, primer extending and        denaturing using a second primer of any one of ACPs described        above having at its 3′-end portion a hybridizing sequence        substantially complementary to a pre-selected sequence at a        second site of the target nucleic acid to hybridize therewith        under conditions in which the second primer anneals to the        second site of the target nucleic acid, whereby a second DNA        strand complementary to the first DNA strand including the        nucleotide variation is generated; and    -   (ii) at least one cycle of primer annealing, primer extending        and denaturing using the same first and second primers as used        in steps (a) and (b)-(i) or a primer pair each having a        hybridizing sequence complementary or corresponding to the 3′-        and 5′-ends of the second DNA strand generated from step (b)-(i)        to hybridize therewith, under conditions in which each primer        anneals to the 3′- and 5′-end sequences of the second DNA        strand, respectively, whereby the second DNA strand which        comprises the first and second sites of the target nucleic acid        at its 3′- and 5′-ends is amplified so that a short target        nucleotide segment corresponding to the second DNA strand        containing the nucleotide variation is generated.

Since this application using the ACP of this invention employs inprinciple the present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity.

A schematic representation of this specific embodiment for singlenucleotide polymorphism (SNP) genotyping using novel ACP is illustratedin FIG. 7A.

The formula of ACP for the detection of a nucleotide variation, isidentical to the formula (1) in which its 3′-end portion comprises ahybridizing sequence substantially complementary to a pre-selectedsequence at a site of the target nucleic acid to hybridize therewithwhich contains the nucleotide variation, wherein the nucleotidecorresponding to the nucleotide variation and its complementarynucleotide of the ACP occupy an interrogation position. The process forthis application is carried out by two stage PCR amplifications usingthe genomic DNA obtained from samples such as patient blood or a shortsegment of the sample DNA, which includes a target nucleotide variation.The interesting nucleotide sample may be obtained from human nucleicacid and an organism that can cause an infectious disease.

The method using two-stage PCR amplifications for detecting singlenucleotide polymorphism (SNP) genotyping basically follows the processused for amplifying a target nucleic acid sequence using genomic DNA asa starting material. In addition, the process for multiplex DNAamplification can be adapted to this application.

To use a short segment of the sample DNA including a target nucleotidevariation as a starting material for the above process, it is preferablethat the target short segment is pre-amplified prior to step (a) using aprimer pair in which each has a hybridizing sequence substantiallycomplementary to the sample DNA to hybridize therewith. Furthermore,more than one target nucleotide segment each including a SNP can beprepared by the multiplex DNA amplification as described in ApplicationII to be used as a starting material in the subject invention formultiple SNP screening.

The first ACP used in step (a) for the detection of a polymorphic baseis an allele-specific ACP which contains an interrogation positionwithin its 3′-end portion occupied by a complementary nucleotide to thecorresponding nucleotide of the nucleotide variation in a target nucleicacid. Preferably, the interrogation position of the first primer is inthe middle of its 3′-end portion. In a more preferred embodiment, theinterrogation position of the allele-specific ACP is within about 10bases of the 3′-end nucleotide. More advantageously, the interrogationposition of the allele-specific ACP is within about 6 bases of the3′-end nucleotide of the allele-specific ACP. In another preferredembodiment, the interrogation position of the allele-specific ACP islocated within positions 4 and 6 from the 3′-end nucleotide. Mostpreferably, the interrogation position of the allele-specific ACP islocated in position 5 from the 3′-end nucleotide. The term “3′-endnucleotide” used herein refers to a nucleotide which is positioned atthe 3′-end of ACP.

In another embodiment, the 3′-end portion of the allele-specific ACPused in step (a) contains at least 6 nucleotides in length, which is aminimal requirement of length for primer annealing. Preferably, the3′-end portion sequence is about 8 to 20 nucleotides in length. Mostpreferably, the 3′-end portion sequence is about 10 nucleotides inlength including an interrogation position.

In one embodiment, at least one artificial mismatch can be also placedwithin the 3′-end portion of ACP using universal base ornon-discriminatory analog that hydrogen-bonds minimally with all fourbases without steric disruption of the DNA duplex. Although the positionof the artificial mismatch can vary depending on experimental designs,it is preferred that the mismatch nucleotide is substantially adjacentthe interrogation position of the first primer.

In a preferred embodiment, the first or second primers comprise at leastone nucleotide with a label for detection or isolation.

According to a preferred embodiment, the first DNA strand includingnucleotide variation in step (a) is generated by one cycle of primerannealing, primer extending, and denaturing. It is preferred that thesecond DNA strand including nucleotide variation in step (b)-(i) isgenerated by one cycle of primer annealing, primer extending, anddenaturing. Preferably, the second DNA strand including nucleotidevariation in step (b)-(ii) is amplified by at least 5 cycles of primerannealing, primer extending, and denaturing.

In another specific embodiment of this invention using amplified shortDNA strand fragment containing the nucleotide variation, there isprovided the method using two individual amplifications of a first and asecond amplifications in which the second amplification is performedusing two stage amplifications, which comprises:

(a) performing the first amplification to produce a short DNA strandfragment containing the nucleotide variation between its ends comprisingat least two cycles of primer annealing, primer extending anddenaturing, using a primer pair each primer comprising a hybridizingsequence substantially complementary to a pre-selected sequence at asite of the target nucleic acid under conditions that the nucleotidevariation is positioned between the pre-selected sequences, in which atleast one primer of the primer set is any one of ACPs described abovehaving at its 3′-end portion the hybridizing sequence, whereby the shortDNA strand fragment containing the nucleotide variation between its endsis amplified;

(b) performing a first-stage amplification of the second amplificationto produce a first DNA strand complementary to the short DNA strandfragment including the nucleotide variation at a first annealingtemperature comprising at least one cycle of primer annealing, primerextending and denaturing, using a first primer of any one of ACPsdescribed above having at its 3′-end portion a hybridizing sequencesubstantially complementary to a pre-selected sequence at a first siteof the target nucleic acid to hybridize therewith, wherein each of thefirst primer and the first site comprises an interrogation positioncorresponding to the nucleotide variation, whereby the first DNA strandcomplementary to the target nucleic acid including the nucleotidevariation is generated when the interrogation position is occupied bythe complementary nucleotide of the first primer to its correspondingnucleotide of the first site; and

(c) performing a second-stage amplification of the second amplificationof the first DNA strand generated from step (a) at a second annealingtemperature, which is high stringent conditions, comprising at least onecycle of primer annealing, primer extending and denaturing using aprimer pair in which amongst the primer pair one is the same as theprimer of any one of ACPs used in step (a) the other is the same as thefirst primer used in step (b), or a primer pair each having ahybridizing sequence complementary or corresponding to the 3′- and5′-ends of the first DNA strand generated from step (b) to hybridizetherewith, under conditions in which each primer anneals to the 3′- and5′-end sequences of the first DNA strand, respectively, whereby thefirst DNA strand is amplified so that a short target nucleotide segmentcorresponding to the first DNA strand containing the nucleotidevariation is generated.

A schematic representation of another specific embodiment for singlenucleotide polymorphism (SNP) genotyping using novel ACP is illustratedin FIG. 7B. Since this specific embodiment is carried out in a similarmanner to above embodiment, the common descriptions between them areomitted in order to avoid the complexity of this specification leadingto undue multiplicity.

The present method can be applied to a variety of nucleotide variationsincluding single nucleotide polymorphism and point mutation(substitution, deletion and insertion).

The amplified products can be analyzed by gel electrophoresis. In oneembodiment, the resulting PCR products can be also detected on adenaturing polyacrylamide gel by autoradiography or non-radioactivedetection methods such as silver staining (Gottschlich et al., 1997;Kociok et al., 1998), the use of fluoresenscent-labelledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), and the use of biotinylated primers (Korn etal., 1992; Tagle et al., 1993; Rosok et al., 1996).

The amplified products generated by multiplex DNA amplification formultiple SNP screening can be compared through the size separation ofthe products. The size separation comparison is also performed byelectrophoresis through an agarose gel matrix or polyacrylamide gelmatrix or sequencing. The products can be also detected by the use offluoresenscent-labelled oligonucleotide primers for automatic analysis.

The term “interrogation position” as used herein refers to the locationof a specific nucleotide base of interest within a target nucleic acid.For example, in the analysis of SNPs, the “interrogation position” inthe target nucleic acid is in position what would be different from wildtype. The interrogation position also includes the location ofnucleotide sequence of a primer which is complementary to aninterrogation position of the target nucleic acid. The interrogationposition of the target nucleic acid is opposite the interrogationposition of the primer, when the primer is hybridized with the targetnucleic acid.

The term “polymorphism” as used herein refers to the presence of two ormore alternative genomic sequences or alleles between or among differentgenomes or individuals. “Polymorphic” refers to the condition in whichtwo or more variants of a specific genomic sequence can be found in apopulation. A “polymorphic site” is the locus at which the variationoccurs. A single nucleotide polymorphism, or SNP, is a single base-pairvariant, typically the substitution of one nucleotide by anothernucleotide at the polymorphic site. Deletion of a single nucleotide orinsertion of a single nucleotide, also give rise to single nucleotidepolymorphisms. Typically, between different genomes or between differentindividuals, the polymorphic site may be occupied by two differentnucleotides. The term “allele” as used herein refers a specific memberof a collection of naturally occurring sequence variants (detectablewithin a population of individuals) at a specific genomic locus ormarker.

In still another aspect of this invention, there is provided a kit foridentifying a nucleotide variation in a target nucleic acid, whichcomprises the annealing control primer or annealing control primer set(including the first and second primers) described above. Thedescriptions of the kits for the amplification of nucleic acid sequenceof this invention can be applied to the present kit.

X. Application to Mutagenesis

This application using ACP of the subject invention can also provide animproved method for mutagenesis. The ACP-based PCR provides an excellenttool for mutagenesis, including deletion, or insertion of sequences, thealteration of one or a few specific nucleotides, and the random mutationof nucleotide sequence.

In further aspect of this invention, there is provided a method formutagenesis in a target nucleic acid, comprising performing anamplification reaction using primers, characterized in that at least oneprimer is derived from any one of ACPs described above. Preferably, theprimer having the structure of ACP is one having at its 3′-end portion ahybridizing sequence substantially complementary to a region of targetnucleic acid sequence, wherein the hybridizing sequence has a nucleotidesequence responsible for mutagenesis.

In a specific embodiment of this invention, there is provided the methodusing two stage amplifications, which comprises:

(a) performing a first-stage amplification of the target nucleic acidsequence at a first annealing temperature comprising at least two cyclesof primer annealing, primer extending and denaturing, using a primerpair of any one of ACPs described above each having at its 3′ endportion a hybridizing sequence substantially complementary to a regionof the target nucleic acid sequence to hybridize therewith, wherein thehybridizing sequence has at least one mismatch nucleotide to generatesite-directed mutation, under conditions in which the primer or primerpair anneals to its target nucleotide sequence, whereby an amplificationproduct containing site-directed mutation site is generated; and

(b) performing a second-stage amplification of the amplification productgenerated from step (a) at a second annealing temperature, which is highstringent conditions, comprising at least one cycle of primer annealing,primer extending and denaturing, using the same primers as used in step(a) or a primer pair each comprising a pre-selected arbitrary nucleotidesequence corresponding to each 5′-end portion of the primers used instep (a), under conditions in which each primer anneals to the 3′- and5′-ends of the amplification product, respectively, whereby theamplification product containing site-directed mutation site isre-amplified.

This specific embodiment relates to site-directed mutagenesis.

Since this application using the ACP of this invention employs inprinciple the present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity.

The formula of ACP for PCR mutagenesis is identical to the formula (1)in which the 3′-end portion comprises a sequence for site-directedmutagenesis or for random mutation.

In still further aspect of this invention, there is provided a kit formutagenesis in a target nucleic acid, which comprises the annealingcontrol primer or annealing control primer set described above. Thedescriptions of the kits for the amplification of nucleic acid sequenceof this invention can be applied to the present kit.

XI. Other Applications

The ACP of the subject invention can be also useful in a variety ofprocesses involving nucleic acid amplifications, particularly, PCR. Forexample, the processes include mixed oligonucleotide-primedamplification of cDNA, long-range PCR, linear PCR, inverse PCR,quantitative PCR, touchdown PCR, sequencing, in situ PCR, vectorette PCRand thermal asymmetric interlaced PCR. The general procedures for thesemethods can be found in Joseph Sambrook, et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001) and M. J. McPherson, et al., PCR, Springer-VerlagNew York Inc., N.Y. (2000).

Therefore, the present invention encompasses all uses of the primeraccording to ACP for process involving nucleic acid amplification,particularly PCR.

The ACP of this invention is significantly effective and widelyaccessible to nucleic acid amplification-based applications. Also,various problems related to primer annealing specificity remained in theprevious nucleic acid amplification techniques can be fundamentallysolved by the ACP and the methods of the present invention. The mainbenefits to be obtained from the use of the ACP during nucleic acidamplification are as follows:

(a) since the presence of a regulator portion positioned between the 3′-and 5′-end portions restricts primer annealing portion to the 3′-endportion under such conditions that the 3′-end portion anneals to thetemplate, the annealing sequence of a primer can be preciselycontrolled, which makes it possible to design a primer caplable ofhaving only a desired number of sequence annealed (or possible to designa primer capable of controlling an annealing portion thereof). It isparticularly useful when an annealing portion of a primer has to belimited (e.g., SNP genotyping, DNA microarrary screening, and detectionof differentially expressed genes);

(b) since the presence of a regulator portion positioned between the 3′-and 5′-end portions interrupts the annealing of the 5′-end portion tothe template under such conditions that the 3′-end portion anneals tothe template, eventually the 5′-end portion not involved in theannealing provides the 3′-end portion with primer annealing specificity;

(c) the specificity of primer annealing is highly sensitive enough todetect even a single-base mismatching. It is particularly useful forsinglenucleotide polymorphisms (SNPs) genotyping;

(d) the ACP is capable of providing a primer with a high tolerance in“primer search parameters” for primer design such as primer length,annealing temperature, GC content, and PCR product length;

(e) the ACP provides two-stage nucleic acid amplifications which allowthe amplified products to be excluded from non-specific amplification;

(f) the efficiency of nucleic acid amplification is increased, whichmakes it easier to detect rare mRNAs; and

(g) the reproducibility of nucleic acid amplification products isincreased, which saves a great amount of time and cost.

As much as the nucleic acid amplification technology such as PCR hasinfluenced the biotechnological field, the use of ACP fundamentallyalter the principles of the current existing nucleic acid amplificationmethods, as mentioned above, by exhibiting unlimited applicability, andhave them significantly upgraded at one time. In consequence, the ACPand its various applications described herein provide a turning point toopen a new biotechnological era since the introduction of nucleic acidamplification technology.

The following specific examples are intended to be illustrative of theinvention and should not be construed as limiting the scope of theinvention as defined by appended claims.

EXAMPLES

In the experimental disclosure which follows, the followingabbreviations apply to: M (molar), mM (millimolar), μM (micromolar), g(gram), μg (micrograms), ng (nanograms), 1 (liters), ml (milliliters),1A1 (microliters), ° C. (degree Centigrade); Promega (Promega Co.,Madison, USA); Clontech (CLONTECH Laboratories, Palo Alto, USA); Roche(Roche Diagnostics, Mannheim, Germany); QIAGEN (QIAGEN GmbH, Hilden,Germany).

The primers used in the subject invention are shown in Table 1.

Example 1 Evaluation of Universal Base Effect in ACP

The effect of universal base residues positioned between the 3′- and5′-end portions of ACP was evaluated by RT-PCR using mouse conceptustissues.

Total RNA was isolated from the entire conceptuses of mouse strain ICRat the day of 4.5, 11.5 and 18.5 during gestation period using eitherTri-reagent (Sigma), or the LiCl/Urea method (Hogan et al., 1994) aspreviously described (Chun et al., 1999; Hwang et al., 2000). Twoindividual experiments of cDNA amplifications using ACP were performedto examine the effect of universal base, particularly, deoxyinosineresidues positioned between the 3′- and 5′-end portions of ACP asfollows: A. The effect of deoxyinosine residues positioned between the3′- and 5′-end portions of ACP in comparison with ACP and theconventional primer not containing a dexoyinosine group; B. The effectof deoxyinosine residues positioned between the 3′- and 5′-end portionsof ACP in association with the alteration of number of dexoyinosine.

These experiments were conducted based on the following assumptions:

(i) the presence of universal base residues which have lower T_(m), thanother portions in ACP due to their weaker hydrogen bonding interactionsin base pairing would not be involved in annealing to the templatenucleic acid under the conditions that the 3′-end portion of ACP annealsto a site of the template at a first annealing temperature.

(ii) the presence of at least one universal base residue between the 3′-and 5′-end portions of ACP would be capable of interrupting theannealing of the 5′-end portion and restricting a primer annealingportion to the 3′-end.

(iii) the 3′-end portion of ACP would act only as a annealing portion tothe template during PCR.

(iv) the 3′-end portion of dT-ACP which is dT₁₀ comprising 10 Tnucleotides also has too low T_(m) to bind the template nucleic acid.

(v) consequently, the dT₁₀-ACP does not produce any PCR products underhigh annealing temperature.

A. The Effect of Deoxyinosine Residues Positioned Between the 3′- and5′-End Portions of ACP in Comparison with ACP with the Primer notContaining a Dexoyinosine Group

(a) First-Strand cDNA Synthesis

dT₁₀-JYC2 5′-GCTTGACTACGATACTGTGC-GATTTTTTTTT-3′ (SEQ ID NO:29) ordT₁₀-ACP1 5′-GCT-TGACTACGATACTGTGCGAIIIIITTTTTTTTTT-3′ (SEQ ID NO: 30)was used as a cDNA synthesis primer.

Three micrograms of total RNA and 2 μl of 10 μM dTIO-JYC2 or 10 μMdT10-ACP1 were combined in a 20 μl final volume. The solution was heatedat 65° C. for 10 minutes, quenched on ice, and microcentrifuged tocollect solvent at the bottom. The following components were addedsequentially to the annealed primer/template on ice: 0.5 μl (40units/μl) of RNasin ribonuclease inhibitor (Promega), 4 μl of 5×reaction buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl₂, 50 mMDTT; Promega), 5 μl of 2 mM each deoxynucleotide mix (dATP, dCTP, dGTP,dTTP), and 1 μl of Moloney-murine leukemia virus (M-MLV) reversetranscriptase (200 units/μl; Promega). The 20 μl of reaction mixture wasincubated at 37° C. for 90 min, microcentrifuged, and placed on ice for2 min. The reaction was stopped by incubation at 94° C. for 2 min.

(b) cDNA Amplification Using ACPs

The dT₁₀-ACP1 was used to examine the effect of a deoxyinosine grouppositioned between the 3′- and 5′-end portions during PCR. The dT₁₀-JYC2not containing a deoxyinosine group was used as a control.

The ACP10 5′-GTCTACCAGGCATTCGCTTCATIIIIIGCCATCGACC-3′ (SEQ ID NO:13) wasused as 5′ primer for this experiment.

The PCR amplification was conducted in a 50 μl volume containing 50 ngof the first-strand cDNA, 5 μl of 10×PCR buffer, 1 μl of 10 μM 5′ primer(ACP10), 1 μl of 10 μM 3′ primer (dT10-JYC2 or dTIO-ACP1), 3 μl of 25 mMMgCl₂, 5 μl of 2 mM dNTP, 0.5 μl of Taq polymerase (5 units/μl). The PCRreactions were conducted under the following conditions: 5 min at 94°C., followed by 30 cycles of 94° C. for 1 min, 54° C. for 1 min, and 72°C. for 1 min; followed by a 5 min final extension at 72° C. Amplifiedproducts were analyzed by electrophoresis in a 2% agarose gel followedby ethidium bromide staining.

As a result, FIG. 8 shows that the dT10-ACP1 containing a deoxyinosinegroup produced almost no products (lanes 4-6), whereas the dT10-JYC2 notcontaining a deoxyinosine group produced a plurality of amplified cDNAproducts (lanes 1-3). Consistent with our assumption, the resultsclearly indicate that the deoxyinosine group positioned between the 3′-and 5′-end portions affects the annealing of the 3′- and 5′-end portionsof the dTIO-ACP to the template cDNA under such high annealingtemperature, resulting in no product as stated in the above assumption.

B. The Effect of Deoxyinosine Residues Positioned Between the 3′- and5′-End Portions of ACP in Association with the Alteration of Number ofDexoyinosine

(a) First-Strand cDNA Synthesis

The first-strand cDNA was synthesized from total RNA of mouse concentuesusing dT10-JYC2 as a cDNA synthesis primer as the above.

(b) cDNA Amplification Using ACPs

This experiment used four ACPs each comprising different number ofdeoxyinosine residues as follows, to examine the effect of deoxyinosineresidues positioned between the 3′- and 5′-end portions in associationwith the alteration of number of deoxyinosine, under a particularstringency conditions. ACP16 5′-GTCTACCAGGCATTCGCTTCATIIGC-CATCGACC-3′(SEQ ID NO:20); ACP17 5′-GTCTACCAGGCATTCGCTTCATIIIIGC-CATCGACC-3′ (SEQID NO:21); ACP18 5′-GTCTACCAGGCATTCGCTTCATII-IIIIGCCATCGACC-3′ (SEQ IDNO:22); ACP19 5′-GTCTACCAGGCATTCGCTTCATII-IIIIIIGCCATCGACC-3′ (SEQ IDNO:23); and CRP210 5′-GTCTACCAGGCATTCGCTTCATGCCATC-GACC-3′ (SEQ IDNO:19) not containing a deoxyinosine group was used as a control.

The resultant first-strand cDNA aenerated from step (A), which comprisesthe preselected arbitrary sequence of the dT10-ACP at its 5′-end, wasused as a template and the primer JYC2 5′-GCTTGACTACGATACTGTGCGA-3′ (SEQID NO:10) corresponding to the 5′-end portion of the dT10-ACP was usedas 3′ primer.

The PCR amplification was conducted in a 50 μl volume containing 50 ngof the first-strand cDNA, 5 μl of 10×PCR buffer, 1 μl of 10 μM 5′ primer(ACP16, 17, 18, 19, or CRP210), 1 μl of 10 μM 3′ primer (JYC2), 3 μl of25 mM MgCl₂, 5 μl of 2 mM dNTP, 0.5 μl of Taq polymerase (5 units/111).The PCR reactions were comprised of: 5 min at 94° C., followed by 30cycles of 94° C. for 1 min, 57° C. for 1 min, and 72° C. for 1 min;followed by a 5 min final extension at 72° C. Amplified products wereanalyzed by electrophoresis in a 2% agarose gel followed by ethidiumbromide staining.

As a result, FIG. 9 shows that the CRP210 not containing anydeoxyinosine residues produced a plurality of amplified cDNA products,whereas the ACPs containing at least two deoxyinosine residues generatedthe significant reduction of amplified cDNA products, and even more, theACP containing eight deoxyinosine residues produced almost no products.Consistent with our assumption, the results clearly indicates that theannealing of the 3′-end portion of ACP to the template could beseparated from the 5′-portion since a group of contiguous deoxyinosineresidues separates the annealing of the 3′-end and 5′-end portions underhigh stringent conditions due to the property of deoxyinosine such asits weaker hydrogen bonding interaction in base pairing.

Example 2 Method for Amplifying a Target Nucleic Acid Sequence Using ACP

The ACP of the subject invention was applied to amplify targetnucleotide sequences of mouse placenta-specific homeobox gene Esx1 cDNA.The process and results for the amplification of the target nucleotidesequences of Esx1 cDNA using ACPs are described herein. Total RNA (3 μg)obtained from mouse 18.5-day-old placenta was used as a startingmaterial. First-strand cDNAs were prepared under the same conditions asused in the cDNA synthesis of Example 1, except that Oligo-dT15 was usedas the first-strand cDNA synthesis primer.

Oligo-dT15 (SEQ ID NO: 54) 5′- TTTTTTTTTTTTTTT-3′

The resultant first-strand cDNAs were used as templates to amplifytarget cDNA fragments of Esx1 using ACPs. These experiments conductedtwo stage PCR amplifications, which is one of unique features of thepresent invention.

The conventional primers of Esx1 used in the Example are:

EsxN7 (SEQ ID NO: 44) 5′-GCCGGTTGCAGAAGCACC-3′; EsxC6 (SEQ ID NO: 45)5′-GAACCATGTTTCTGAATGCC-3′; EsxN1 (SEQ ID NO: 48)5′-GAATCTGAAACAACTTTCTA-3′; EsxC2 (SEQ ID NO: 49)5′-GATGCATGGGACGAGGCACC-3′; EsxN3 (SEQ ID NO: 51)5′-CGCCGCAACCCCTGCCCGCA-3′;  and EsxC5 (SEQ ID NO: 52)5′-GATGCATGGGACGAGGGA-3′

Three primer sets, EsxN7 and EsxC6, EsxN1 and EsxC2, and EsxN3 andEsxC5, were used in the Example because they are known as the primersets which generate high backgrounds as well as non-specific products inconventional PCR methods as known in the art.

According to single-target PCR systems, primers with similar meltingtemperatures (Tm) should be chosen. However, a primer set of EsxN1 (Tm50.7° C.) and EsxC2 (Tm 71.9° C.) shows about 20° C. of differentmelting temperatures between them, and a primer set of EsxN3 (Tm 86.9°C.) and EsxC5 (Tm 66.2° C.) both has high melting temperatures. Also, aprimer set of EsxN7 (Tm 68.2° C.) and EsxC6 (Tm 61.2° C.), which hasrelatively similar melting temperature, are selected to observe theeffect of ACP.

The ACP of the subject invention was applied to these three conventionalprimer sets to demonstrate if the ACP system can overcome the mainproblems arising from these conventional primer sets, such as backgroundand non-specific products.

The following ACPs comprise the sequences of the above conventionalprimers at their 3′-end portions and were used as Esx1 gene-specificprimers for the first-stage PCR amplification:

EsxN7-ACP 5′ primer (SEQ ID NO: 46)5′- GTCTACCAGGCATTCGCTTCATIIIIIGCCGGT TGCAGAAGC ACC-3′; EsxC6-ACP 3′primer (SEQ ID NO: 47) 5′- GCTTGACTACGATACTGTGCGAIIIIIGAACCATGTTTCTGAATGCC-3; EsxN1-ACP 5′ primer (SEQ ID NO: 50)5′- GTCTACCAGGCATTCGCTTCATIIIIIGAATCT GAAACAACT TTCTA-3′; EsxC2-ACP 3′primer (SEQ ID NO: 55) 5′-GCTTGACTACGATACTGTGCGAIIIIIGATGCATGGGACGAG GCACC-3′; EsxN3-ACP 5′ primer (SEQ ID NO: 53)5′-GTCTACCAGGCATTCGCTTCATIIIIICGCCGC AACCCCTG CCCGCA-3′; andEsxC5-ACP 3′ primer (SEQ ID NO: 56)5′-GC′TTGACTACGATACTGTGCGAIIIIIGATGCA TGGGACGA GGCA-3′

The 5′-end portion sequences of the ACPs were served as pre-selectedarbitrary primer sequences only for the second-stage PCR amplification:JYC2 and JYC4 5′-GTCTACCAGGCATTCGCTTCAT-3′ (SEQ ID NO:12).

During the first-stage PCR amplification, the primer set of EsxN7-ACPand EsxC6-ACP was used as 5′ and 3′ primers, respectively, to generatethe 520-bp fragment of the Esx 1 cDNA, the primer set of EsxN1-ACP andEsxC2-ACP was used as 5′ and 3′ primers, respectively, to generate the784-bp fragment of the Esx1 cDNA, and the primer set of EsxN3-ACP andEsxC5-ACP was used as 5′ and 3′ primers, respectively, to generate the483-bp fragment of the Esx1 cDNA.

During the second-stage PCR amplification, JYC4 and JYC2 were used aspreselected arbitrary 5′ and 3′ primers, respectively (PROTOCOL A). Asan alternative, the complete sequences of the ACPs, instead of thepre-selected arbitrary primers such as JYC4 and JYC2, can be used as 5′and 3′ primers for the second-stage PCR amplification at the highstringent conditions. In this case, it is not necessary to add thepre-selected arbitrary primers to the reaction mixture at the time of orafter the first-stage PCR reaction (PROTOCOL B).

Protocol A: One-Stop Two-Stage PCR Amplifications

(A) First-Stage PCR Amplification

The first-stage PCR amplification was performed by hot start PCR methodin which the procedure is to set up the complete reactions without theDNA polymerase and incubate the tubes in the thermal cycler to completethe initial denaturation step at >90° C. Then, while holding the tubesat a temperature above 70° C., the appropriate amount of DNA polymerasecan be pipetted into the reaction.

The first-stage PCR amplification was conducted by two cycles of PCRconsisting of annealing, extending and denaturing reaction; the reactionmixture in a final volume of 49.5 μi containing 50 ng of thefirst-strand cDNA, 5 μl of 10×PCR reaction buffer (Promega), 5 μl of 25mM MgCl₂, 5 μl of dNTP (2 mM each dATP, dCTP, dGTP, dTTP), 1.35 μl of 5′ACP (1 μM) and 1.35 μl of 3′ ACP (1 μM) is pre-heated at 94° C., whileholding the tube containing the reaction mixture at the 94° C., 0.5 μlof Taq polymerase (5 units/μl; Promega) is added into the reactionmixture; the PCR reactions comprise two cycles of 94° C. for 40 sec, 60°C. for 40 sec, and 72° C. for 40 sec; followed by denaturing theamplification product at 94° C.

(B) Second-Stage PCR Amplification

The resultant cDNA product generated by the first-stage PCRamplification using Esx1 gene-specific ACPs was then amplified by thefollowing second-stage PCR amplification under higher annealingtemperature. After the completion of the first-stage PCR amplification,each 1 μl of 10 μM pre-selected arbitrary primers, JYC4 and JYC2, wasadded into the reaction mixture obtained from the first-stage PCRamplification, under denaturing temperature such as at 94° C. The secondstage-PCR reaction was as follows: 35 cycles of 94° C. for 40 sec, 68°C. for 40 sec, and 72° C. for 40 sec; followed by a 5 min finalextension at 72° C.

The amplified products were analyzed by electrophoresis in a 2% agarosegel and detected by staining with ethidium bromide. The resulting PCRproducts can be also detected on a denaturing polyacrylamide gel byautoradiography or non-radioactive detection methods such as silverstaining (Gottschlich et al., 1997; Kociok et al., 1998), the use offluoresenscent-labelled oligonucleotides (Bauer et al. 1993; Ito et al.1994; Luehrsen et al., 1997; Smith et al., 1997), and the use ofbiotinylated primers (Korn et al., 1992; Tagle et al., 1993; Rosok etal., 1996).

As shown in FIGS. 10A-C, the one-stop two-stage PCR amplifications forEsx 1 using each primer set of EsxN7-ACP and EsxC6-ACP, EsxN1-ACP andEsxC2-ACP, and EsxN3-ACP and EsxC5-ACP generated a single band whichcorresponds to the expected size, 520-bp (FIG. 10A, lane 2), 784-bp(FIG. 10B, lane 4), and 483-bp (FIG. 10C, lane 3) of Esx1 cDNAfragments, respectively. Subsequent cloning and sequence analysis of theclones confirm that the band is Esx1 cDNA fragments. In contrast, theconventional primer sets, which contain the sequences corresponding onlyto the 3′-end portions of each ACP sets, produced non-specific productsas well as high backgrounds such as DNA smear (FIG. 10A, lane 1; FIG.10B, lane 3; FIG. 10C, lanes 1 and 2). Since the PCR products using aACP set comprise the pre-selected arbitrary sequences at their 5′- and3′-ends, additional 54-bp sequences corresponding to the pre-selectedarbitrary sequences and deoxyinosine residues were found.

FIG. 10A shows the amplified cDNA products generated by the followingsets of primers; a set of EsxN7 and EsxC6 (lane 1), and a set ofEsxN7-ACP and EsxC6-ACP (lane 2). PCR reactions using the conventionalprimer set EsxN7 and EsxC6 were as follows: 5 min at 94° C. followed by30 cycles of 94° C. for 40 sec, 60° C. for 40 sec, and 72° C. for 40sec; followed by a 5 min final extension at 72° C.

FIG. 10B shows the amplified cDNA products generated by a single primeror a primer pair as follows: the primers, EsxN1 and EsxC2, were used inlanes 1 and 2, respectively; a combination of EsxN1-ACP and conventionalprimer EsxC2 was used in lanes 3; two ACPs EsxN1-ACP and EsxC2-ACP wereused in lane 4. When a conventional primer set, EsxN1 and EsxC2, wasused under high annealing temperature of 60° C., no specific-targetproduct was produced. When a primer set comprising one ACP EsxN1-ACP anda conventional primer of EsxC2 was used, a target-specific product aswell as nonspecific products were amplified due to the non-specificbinding of the conventional primer EsxC2 (lane 3). However, when a ACPset was used, only a single target-specific product was amplified (lane4), which indicates that the ACP of the subject invention providesprimers with tolerance to “primer design parameter” related to meltingtemperatures of general primers requested for single-target PCR systems.

FIG. 10C shows the amplified cDNA products generated by using thefollowing primer sets: a set of EsxN3 and EsxC5 was used in lanes 1 and2, and a set of EsxN3-ACP and EsxC5-ACP was used in lane 3. PCRreactions using the conventional primer set of EsxN3 and EsxC5 were asfollows: 5 min at 94° C. followed by 30 cycles of 94° C. for 40 sec, 58°C. for 40 sec, and 72° C. for 40 sec; followed by a 5 min finalextension at 72° C. (lane 1). The conventional primer set was alsocompared with the ACP set by conducting the same two stage PCRamplifications as used in the ACP, such that its annealing temperatureis increased from 60° C. to 68° C. (lane 2). These results also indicatethat although the conventional primers including ones having high Tm areused in the same two stage PCR amplification, they could not be freefrom the problems of non-specific products and background, whereas theACP of the subject invention can help overcome such problems arisingfrom these conventional primers.

Protocol B: Non-Stop Two-Stage PCR Amplifications

Alternatively, the complete sequences of the ACPs, instead of thepre-selected arbitrary primers such as JYC4 and JYC2, can be used asprimers for the second-stage PCR amplification at the high stringentconditions. In this case, it is not necessary to add the preselectedarbitrary primers to the reaction mixture at the time of or after thefirst-stage PCR reaction.

The process of the non-stop two-stage PCR amplifications is basicallyidentical to Protocol A, except that the ACPs, 1 μl of 5′ ACP (10 μM)and 1 μl of 3′ ACP (10 μM), are added at the first stage PCRamplification and the second stage PCR amplification immediately followsthe first stage PCR amplification without any delay because there is nostep of adding pre-selected arbitrary primers.

The amplified products were analyzed by electrophoresis in a 2% agarosegel and detected by staining with ethidium bromide. The resulting PCRproducts can be also detected on a denaturing polyacrylamide gel byautoradiography or non-radioactive detection methods such as silverstaining (Gottschlich et al., 1997; Kociok et al., 1998), the use offluoresenscent-labelled oligonucleotides (Bauer et al. 1993; Ito et al.1994; Luehrsen et al., 1997; Smith et al., 1997), and the use ofbiotinylated primers (Korn et al., 1992; Tagle et al., 1993; Rosok etal., 1996).

FIG. 10D shows the amplified cDNA products generated by the non-stoptwo-stage PCR Amplifications using the following single primer or aprimer pair; the primers EsxN1 and EsxC2 were used in lane 1 and 2,respectively; a pair of EsxN1 and EsxC2 was used in lane 3; and a pairof EsxN1-ACP and EsxC2-ACP was used in lane 4. When a conventionalprimer set, EsxN1 and EsxC2, was used, no specific-target product wasproduced. However, when a ACP set was used in non-stop two-stage PCRamplifications, only a single target-specific product was amplified(lane 4), which is consistent with the results of one-stop two-stage PCRAmplifications (FIG. 10B).

These examples illustrate that the ACP permits the products to be freefrom the background problems as well as non-specificity arising from theconventional primers used in PCR methods as described in the art. Itcould be also understood that the ACP allows the generation of thespecific products regardless of the design of gene-specific primers.

Example 3 Identification and Characterization of DifferentiallyExpressed mRNAs During Mouse Embryonic Development Using ACP

The ACP of the subject invention has been applied to detectdifferentially expressed mRNAs in embryonic developments. Specifically,three different procedures and results using different stages ofconceptus total RNAs as starting materials are described herein. Theprimers used in the subject invention are shown in Table 1.

A 1. Procedure 1

Step (1): First-Strand cDNA Synthesis

The first-strand cDNAs were prepared under the same conditions as usedin the cDNA synthesis of Example 1 using the dT10-ACP1 or JYC5-T15-ACPas a cDNA synthesis primer. The resultant cDNAs were purified by a spincolumn (PCR purification Kit, QIAGEN) to remove primers, dNTP, and theabove reagents. It is necessary to perform the purification step priorto the determination of the cDNAs concentration using the UVspectroscopy at an absorbance of 260 nm. The same amount of cDNAs fromeach sample was used for comparing their amplification patterns usingthe ACP system described herein,

Step (2): First-Stage PCR Amplification Using ACP

The following ACPs were used as arbitrary ACPs (AR-ACPs) for the firstPCR amplification:

ACP3 (SEQ ID NO :3) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGCCATC GACS-3′; ACP5(SEQ ID NO: 5) 5′-GTCTACCAGGCATTCGCTTCATIIIIIAGGCGA  TGCS-3′; ACP8(SEQ ID NO: 8) 5′-GTCTACCAGGCATTCGCTTCATIIIIICTCCGA  TGCS-3′; ACP10(SEQ ID NO: 13) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGCCATC  GACC-3′; ACP13(SEQ ID NO: 16) 5′-GTCTACCAGGCATTCGCTTCATIIIIIAGGCGA TGCG-3′; and ACP14(SEQ ID NO: 17) 5′-GTCTACCAGGCATTCGCTTCATIIIIICTCCGA  TGCC-3′

The 5′-end portion sequences of the dT10-ACP1 and AR-ACPs serve aspre-selected arbitrary primer sequences only for the second-PCRamplification. The pre-selected arbitrary primers are JYC2 and JYC4.

The first-strand cDNAs produced from step (1) were amplified by thefollowing first-stage PCR amplification using one of AR-ACPs (ACP3,ACP5, ACP8, ACP10, ACP13, or ACP14) and the dT10-ACP1 as 5′ and 3′primers, respectively. The first-stage PCR amplification was conductedin a 50 μl volume containing 50 ng of the first-strand cDNA, 5 μl of10×PCR reaction buffer (Promega), 3 μl of 25 mM MgCl₂, 5 μl of dNTP (0.2mM each dATP, dCTP, dGTP, dTTP), 5 μl of 5′ primer (1 gM), 5 μl of 3′primer (1 μM), and 0.5 μl of Taq polymerase (5 units/μl; Promega). ThePCR reactions were as follows: 5 min at 94° C. followed by 20 cycles of94° C. for 1 min, 50° C. for 1 min, and 72° C. for 1 min; followed by a5 min final extension at 72° C.

The cycle of the first-stage PCR amplification can be varied dependingon the types of samples. For example, the 20 cycles of the first PCRamplification were used for mouse conceptus samples.

Step (3): Second-Stage PCR Amplification Using Pre-Selected ArbitraryPrimers Corresponding to the 5′-End Portion Sequences of ACPs

The amplified cDNA products produced from step (2) are re-amplified bythe following second-stage PCR amplification using two pre-selectedarbitrary primers, JYC4 and JYC2, each corresponding to the 5′-endportion sequences of AR-ACP and dT10-ACP1, respectively. Thesecond-stage PCR amplification was conducted in a 50 μl volumecontaining 5 μl of the first amplified cDNA products (50 μl), 5 μml of10×PCR reaction buffer (Promega), 3 μl of 25 mM MgCl₂, 5 μl of 2 mMdNTP, 1 μl of 5′ primer (10 gM), 1 μl of 3′ primer (10 μM), and 0.5 μlof Taq polymerase (5 units/μl). The PCR reactions were as follows: 5 minat 94° C. followed by 30 cycles of 94° C. for 1 min, 65° C. for 1 min,and 72° C. for 1 min; followed by a 5 min final extension at 72° C.

A2. Procedure 2

The alternative procedure comprises the following steps of:

(a) providing a first sample of nucleic acids representing a firstpopulation of mRNA transcripts and a second sample of nucleic acidsrepresenting a second population of mRNA transcripts;

(b) contacting each of the first nucleic acid sample and the secondnucleic acid sample with a first ACP, wherein the first ACP has ahybridizing sequence substantially complementary to a region of thefirst and second population of mRNA transcripts to hybridize therewith;

(c) reverse transcribing the mRNA to which the first ACP hybridizes toproduce a first population of DNA strands that are complementary to themRNAs in the first nucleic acid sample to which the first ACPhybridizes, and a second population of DNA strands that arecomplementary to the mRNA in the second nucleic acid sample to which thefirst ACP hybridizes;

(d) purifying and quantifying the complementary DNA strands produced asa result of the reverse transcription step (c);

(e) synthesizing a second DNA strand complementary to each of the firstand second populations of DNA strands using a second ACP under lowstringent conditions, by at least one PCR cycle comprising denaturing,annealing and primer extension, wherein the second ACP has a hybridizingsequence substantially complementary to the first and second populationsof DNA strands;

(f) amplifying each second DNA strand obtained from step (e) under highstringent conditions, by at least one PCR cycle comprising denaturing,annealing and primer extension to generate first and second populationsof amplification products using two preselected arbitrary primers eachcomprising a sequence corresponding to each 5′-end portion of the firstand second annealing control primers; and

(g) comparing the amount of individual amplification products in thefirst and second populations of amplification products.

The first-strand cDNAs are synthesized using JYC5-T15-ACP5′-CTGTGAATGCTGC GACTACGATIIIIII-3′ (SEQ ID NO: 61).

The 5′-end portion sequence of the JYC5-T15-ACP serves as a 3′pre-selected arbitrary primer sequence to be used only for the secondstage of PCR amplification:

JYC5 (SEQ ID NO: 60) 5′-CTGTGAATGCTGCGACTACGAT-3′.

Step (1): First-Strand cDNA Synthesis

1. Combine 3 μg total RNA and 2 μl of 10 μM JYC5-T15-ACP in a sterile0.2 ml microcentrifuge tube.

2. Add sterile H2O to a final volume of 9.5 μl. Mix contents and spinthe tube briefly in a microcentrifuge.

3. Incubate the tube at 80° C. for 3 minutes or use a thermocycler forthe same purpose.

4. Cool the tube on ice for 2 minutes. Spin down the contents of thetube briefly in a microcentrifuge.

5. To the same reaction tube add the following reagents: 4 μl 5×First-strand buffer (Promega), 5 μl dNTP (2 mM each dATP, dCTP, dGTP,dTTP), 0.5 μl RNasin inhibitor (40 units/μl, Promega) and 1 μl M-MLVreverse transcriptase (200 U/μl).

6. Mix contents and spin the tube briefly in a microcentrifuge.

7. Incubate the tube at 42° C. for 90 min.

8. Incubate the tube at 94° C. for 2 minutes to terminate first-strandsynthesis.

9. Place the tube on ice for 2 min.

10. Purify the resultant cDNAs by a spin column (PCR purification Kit,QIAGEN) to remove primers, dNTP, and the above reagents.

11. Next, measure the concentration of the cDNAs using the UVspectroscopy at an absorbance of 260 nm.

12. Process to step 2.

Step (2): Second-Strand cDNA Synthesis Using ACP

The same amount of cDNAs from each sample was used for the comparison oftheir amplification patterns using the ACPs described herein. Thesecond-strand cDNA was synthesized using arbitrary ACP10 by hot startPCR method in which the procedure is to set up the complete reactionswithout the DNA polymerase and incubate the tubes in the thermal cyclerto complete the initial denaturation step at >90° C. Then, while holdingthe tubes at a temperature above 90° C., the appropriate amount of DNA,polymerase can be pipetted into the reaction.

1. Combine the following reagents in a sterile 0.2 ml microcentrifugetube: 49.5 μl of the total volume containing 1 μl of first-strand cDNA(50 ng/μl) prepared by step 1, 5 μl of 10×PCR buffer (Roche), 5 μl of 2mM dNTP, 1 μl of 10 μM arbitrary ACP (5′ primer) and 37.5 μl of steriledH2O.

2. Mix contents and spin the tube briefly in a microcentrifuge.

3. Place the tube in the preheated thermal cycler at 94° C.

4. Add the 0.5 μl of Taq polymerase (5 units/μl; Roche) into thereaction, while holding the tube at the temperature 94° C.

5. Conduct PCR reaction under the following conditions: one cycle of 94°C. for 5 min, 50° C. for 3 min, and 72° C. for 1 min; followed bydenaturing the first amplification product at 94° C.

Step (3): PCR Amplification of the Second-Strand cDNAs UsingPre-Selected Arbitrary Primers Corresponding to the 5′-End PortionSequences of ACPs

1. After the completion of the first stage PCR amplification, whileholding the tubes at a temperature above 94° C., add 2 μl of 10 μM JYC4and 2 μl of 10 mM JYC5, in which each corresponds to the 5′-end portionsequences of both 5′ and 3′ ACPs, respectively, into the reactionmixture used in step (2).

2. Conduct second stage PCR reactions under the following conditions: 40cycles of 94° C. for 40 sec, 68° C. for 40 sec, and 72° C. for 40 sec;followed by a 5 min final extension at 72° C.

A3. Procedure 3

As an alternative process, in the step (f) of PROCEDURE 2 the completesequences of the first and second ACPs used in the steps (b) and (e) ofPROCEDURE 2, instead of the pre-selected arbitrary sequences of the5′-end portions of the first and second ACPs, can be used as 3′ and 5′primers, respectively, at the high stringent conditions for amplifyingeach second DNA strand obtained from the step (e) of PROCEDURE 2,wherein the 3′- and 5′-ends of the second DNA strands which wereinitially synthesized using the second ACP comprise the sequence of thefirst ACP and the complementary sequence of the second ACP,respectively, and also serve as perfect pairing sites to the first andsecond ACPs. In this case, it is not necessary to add the pre-selectedarbitrary primers to the reaction mixture at the time of or afterfirst-stage PCR reaction.

Step (1): First-Strand cDNA Synthesis

The first-strand cDNAs were prepared under the same conditions as usedin the cDNA synthesis of PROCEDURE 2 using the JYC5-T15-ACP as a cDNAsynthesis primer.

Step (2): Second-Strand cDNA Synthesis and Amplification Using Non-StopTwo-Stage PCR

The same amount of cDNAs from each sample was used for the comparison oftheir amplification patterns using the ACPs described herein. Thesecond-strand cDNA was synthesized using arbitrary ACP10 by hot startPCR method in which the procedure is to set up the complete reactionswithout the DNA polymerase and incubate the tubes in the thermal cyclerto complete the initial denaturation step at >90° C. Then, while holdingthe tubes at a temperature above 90° C., the appropriate amount of DNApolymerase can be pipetted into the reaction.

1. Combine the following reagents in a sterile 0.2 ml microcentrifugetube: 49.5 μl of the total volume containing 1 μl of first-strand cDNA(50 ng/μl) prepared by step 1, 5 μl of 10×PCR buffer (Roche), 5 μl of 2mM dNTP, 1 μl of 10 μM arbitrary ACP10 (5′ primer), 1 μl of 10 μMJYC5-T15-ACP (3′ primer) and 36.5 μl of sterile dH2O.

2. Mix contents and spin the tube briefly in a microcentrifuge.

3. Place the tube in the preheated thermal cycler at 94° C.

4. Add the 0.5 μl of Tag polymerase (5 units/μl; Roche) into thereaction, while holding the tube at the temperature 94° C.

5. Conduct PCR reaction under the following conditions: one cycle of 94°C. for 1 min, 50° C. for 3 min, and 72° C. for 1 min; followed by 40cycles of 94° C. for 40 sec, 65° C. for 40 sec, and 72° C. for 40 sec;and followed by a 5 min final extension at 72° C.

B. Separation of Amplified PCR Products by Electrophoresis Analysis andRecovery of the Differentially Displayed Bands

The amplified products were analyzed by electrophoresis in a 2% agarosegel and detected by staining with ethidium bromide. Several major bandsdifferentially expressed during embryonic development (E4.5, E11.5, andE18.5) were selected, excised and extracted from the gels usingGENECLEAN II Kit (BIO 101). The resulting PCR products can be alsodetected on a denaturing polyacrylamide gel by autoradiography ornon-radioactive detection methods such as silver staining (Gottschlichet al., 1997; Kociok et al., 1998), the use of fluoresenscent-labelledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), and the use of biotinylated primers (Korn etal., 1992; Tagle et al., 1993; Rosok et al., 1996).

C. Re-Amplification of the Recovered Bands

The bands obtained from step B were re-amplified using the samepre-selected arbitrary primers and PCR conditions as used in PROCEDURE1, 2 and 3.

D. Cloning and Sequencing of the Re-Amplified Fragments

Each amplified fragment was cloned into the pGEM-T Easy vector (Promega)and sequenced with the ABI PRISM 310 Genetic Analyzer (Perkin ElmerBiosystem) using BigDye Terminator cycle sequencing kit (Perkin Elmer).Computer-assisted sequence analysis was carried out using the BLASTsearch program (Basic Local Alignment Search Tool).

E. Northern Analysis

Twenty micrograms of total RNA from conceptus tissues were resolved ondenaturing 1% agarose gels containing formaldehyde, transferred ontonylon membranes (Hybond-N, Amersham, USA), and hybridized with a32P-labeled subcloned PCR product in QuikHyb solution (Stratagene, USA)overnight at 58° C. as previously described (Chun et al., 1999; Hwang etal., 2000). Blots were washed at 65° C. twice for 20 min in 2×SSC, 0.1%SDS, twice for 20 min in 1×SSC, 0.1% SDS, and twice for 20 min in0.1×SSC, 0.1% SDS. The membranes were exposed to Kodak X-Omat XK-1 filmwith a Fuji intensifying screen at −80° C.

FIGS. 11A-D shows the amplified cDNA products, wherein mouse conceptussamples obtained from different stages were amplified by PROCEDURE 1using the primer sets as follows; a set of ACP3 and dT10-ACP1 for thelanes 1-3 of FIG. 11A; a set of ACP5 and dT10-ACP1 for the lanes 1-6 andof FIG. 11B and a set of ACP8 and dT10-ACP1 for the lanes 7-12 of FIG.10B, respectively. FIG. 11B also shows additional results of theamplified cDNA products generated by using another ACP sets. FIGS. 11C-Dshows the amplified products generated by using two primer sets of theACP10 and dTIO-ACP1 (FIG. 11C), and ACP14 and dT10-ACP1 (FIG. 11D),respectively. Many differentially expressed bands in a specific stagewere obtained, subcloned into the pGEM-T Easy vector (Promega), andsequenced. Sequence analysis reveals that all of the clones are knowngenes except two novel genes (Table 2). The expression patterns wereconfirmed by Northern blot analysis using mouse conceptus stage blot(Seegene, Inc., Seoul, Korea).

FIG. 12A shows the amplified cDNA products, wherein mouse conceptussamples (E4.5: lane 1; E11.5: lane 2; E18.5: lane 3) obtained fromdifferent stages were amplified by PROCEDURE 2 using a set of ACP10 andJYC5-T15-ACP. Many differentially expressed bands in a specific stagewere obtained, subcloned into the pGEMT Easy vector (Promega), andsequenced. Sequence analysis reveals that all of the clones are knowngenes except one DEG 2 (Table 2). The expression patterns were confirmedby Northern blot analysis using mouse conceptus stage blot (Seegene,Inc., Seoul, Korea).

FIG. 12B shows the amplified cDNA products, wherein mouse conceptussamples at the different stages of (E4.5: lane 3; E11.5: lane 4; E18.5:lane 5) were amplified by non-stop two-stage PCR amplifications using aset of ACP10 and JYC5-T15-ACP as above mentioned in PROCEDURE 3. When aset of ACP10 and JYC5-T15-ACP (lanes 35) was used, the resultant bandswere identical to the bands which were obtained by PROCEDURE 2comprising one-stop two-stage PCR amplifications (FIG. 12A). However, noproducts were generated when a single primer, ACP10 (lane 1) orJYC5-T15-ACP (lane 2) was used, which indicates that the amplifiedproducts were generated only when both ACP 10 and JYC5-T15-ACP as a setwere used for their specific bindings.

FIG. 13 shows the results of Northern blot hybridization forrepresenting six different clones using DEG1 (A; arrow 1 of FIG. 10A,FIG. 11, and FIG. 12), DEG2 (C), DEG3 (B; arrow 2 of FIG. 10A, FIG. 11,and FIG. 12), DEG5 (E), DEG7 (F), and DEG8 (D; arrow 4 of FIG. 10A, FIG.11, and FIG. 12) as probes. The DEG1 probe was also hybridized to thealternative isoform of Tropomyosin 2 (arrow 1′ of FIG. 11, and FIG. 12),which was discovered by this present invention. Consistent with theresults of agarose gel analysis, Northern blot analysis showed that theexpression patterns of the clones are identical to the original bands onthe agarose gels, indicating that all of the clones are true positiveproducts. Thus, the ACP produces only positive products without anyfalse positives, which means that the ACP eliminates the problem offalse positives.

FIG. 14 shows the results of Northern blot hybridization for theexpression of DEG5 during mouse embryonic development. DEG5, which isturned out as a novel gene by sequence analysis, shows an interestingexpression patterns: after a strong expression appeared in the earlypregnancy stage (E4.5), its expression was gradually reduced in themiddle stages and gradually increased again in the late developmentstage (E17.5 and E18.5).

These results indicate that the method using ACP for isolatingdifferentially expressed genes produces only real PCR products andcompletely eliminates false positive products. Freedom from falsepositives which have been one major bottleneck remaining for theprevious Differential Display technique allows avoiding the subsequentlabor-intensive work required for the verification of the cDNA fragmentsidentified by Differential Display.

Example 5 Method for Rapid Amplification of 3′-Ends of cDNA (3′-RACE)Using ACP

The present example compares the ACP-based 3′-RACE and the conventional3′-RACE in order to demonstrate if the ACP of the present invention canexclude such background problems arising from conventional oligo-dTprimers used in cDNA synthesis.

In the conventional 3′-RACE, the poly(A) tail of mRNA molecules isexploited as a priming site for PCR amplification and thus the oligo-dTprimer is used as a 3′ primer for the conventional 3′-RACE. In contrast,the ACP of the present invention uses the poly(A) tail of mRNA as apriming site only for the cDNA synthesis but not for the subsequent PCRamplification.

Mouse first-strand cDNAs were prepared under the same conditions as usedin the cDNA synthesis of Example 1 using Oligo VdT15-ACP5′-GCTTGACTACGATACTGTGC-GAIIIIITTTTTTTTTTTTTTTV-3′ (SEQ ID NO:57) (V isA, C or G) as a cDNA synthesis primer and then, directly used astemplates for the subsequent PCR amplification without the purificationstep for the removal of the cDNA synthesis primer.

For the conventional 3′-RACE, the first-strand cDNAs were synthesizedusing the following cDNA synthesis primer;

CDS 111/3′ (SEQ ID NO: 35) 5′-ATTCTAGAGGCCGAGGCGGCCGACATG-(dT)30-VN-3′(V is A, C or G; and N is A, C, T or G).

This cDNA synthesis primer, CDS 111/3′, was used as 3′ primer forsubsequent PCR amplification.

The PCR amplification was conducted in a 50 μl volume containing 50 ngof the first-strand cDNA, 5 μl of 10×PCR buffer (Promega), 1 μl of agene-specific 5′ primer (10 μM), 1 μl of pre-selected arbitrary 3′primer JYC2 (10 μM) or CDS III/3′ (10 μM), 3 μl of 25 mM MgCl₂, 5 μl of2 mM dNTP, 0.5 μl Taq polymerase (5 units/μl; Promega). The PCRreactions were conducted under the following conditions: 5 min at 94° C.followed by 30 cycles of 94° C. for 1 min, 65° C. for 1 min, and 72° C.for 1 min; followed by a 5 min final extension at 72° C. Amplifiedproducts were analyzed by electrophoresis in a 2% agarose gel followedby ethidium bromide staining. The resulting PCR products can be alsodetected on a denaturing polyacrylamide gel by autoradiography ornon-radioactive detection methods such as silver staining (Gottschlichet al., 1997; Kociok et al., 1998), the use of fluoresenscent-labelledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), and the use of biotinylated primers (Korn etal., 1992; Tagle et al., 1993; Rosok et al., 1996).

FIG. 15 shows the results of beta-actin 3′-RACE. The conventional3′-RACE (lane 1) was compared with ACP-based 3′-RACE (lane 2). Theconventional 3′-RACE method produced non-specific products as well asDNA smear background, whereas the ACP-based 3′-RACE produced only asingle band, which is the expected size of 348-bp. These resultsindicate that the ACP-based 3′-RACE can exclude the background problemssuch as DNA smear and non-specific products.

Example 6 Method for Rapid Amplification of 5′-End (5′-RACE) andFull-Length cDNAs Using ACP

The ACP of the subject invention was also used to amplify the 5′-ends ofcDNA fragments. The first-strand cDNAs were synthesized using OligoVdT15-ACP, or Random dN6-ACP:

Oligo VdT₁₅-ACP (SEQ ID NO: 57) 5′-GCTTGACTACGATA CTGTGCGAIIIIITTTTTT TTTTTTTTTV-3′, wherein V can be A, C, or G; Random dN₆-ACP(SEQ ID NO: 58) 5′-GCTTGACTACGATACTGTGCGAIIIIINNNNN N-3′,wherein N can be A, C, G, or T.

After the complete synthesis of the first strand cDNA sequences presentin the form of mRNA-cDNA intermediates, cytosine residues are tailed atthe 3′-end of the first strand cDNA sequences by the terminaltransferase reaction of reverse transcriptase in the presence ofmanganese. The 3′-ends of the first strand cDNAs were extended using thefirst strand cDNA 3′-end extending ACP (rG3-ACP, rG2-ACP, or dG3-ACP)and then, directly used as templates for the subsequent PCRamplification without a purification step for the removal of the firststrand cDNA 3′-end extending ACP as well as the cDNA synthesis primer.

The sequences of the first-strand cDNA 3′-end extending ACPs are:

rG3-ACP (SEQ ID NO: 36) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGGr(GGG)-3′; rG2-ACP (SEQ ID NO: 37) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGGr(GG)-dG-3′; rG1-ACP (SEQ ID NO: 59) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGGr(G)-d(GG)-3′; or dG3-ACP (SEQ ID NO: 38)5′-GTCTACCAGGCATTCGCTTCATIIIIIGGd  (GGG)-3′(wherein r and d represent ribonucleotide  and deoxyribonucleotide, respectively).A. First-Strand Full-Length cDNA SynthesisProtocol A: First-Strand cDNA Synthesis Using the ACP of the SubjectInvention

1. Combine the followings in a sterile 0.2 ml microcentrifuge tube: 3 μgof total RNA and 2 μl of 10 μm of Oligo VdT15-ACP or random dN6-ACP.

2. Add sterile H2O to a final volume of 10 μl. Mix contents and spin thetube briefly in a microcentrifuge.

3. Incubate the tube in a 65° C. water bath for 15 minutes or use athermocycler for the same purpose.

4. Cool the tube on ice for at least 2 minutes. Spin down the contentsof the tube briefly in a microcentrifuge.

5. Add the following reagents to the same reaction tube: 4 μl of 5×first-strand buffer (Invitrogen), 1 μl of 0.1 M DTT, 2 μl of BSA (1mg/ml), 2 μl of dNTP (10 mM each dATP, dCTP, dGTP, dTTP), 0.4 μl of 100mM MnCl2 and 0.5 μl of RNasin inhibitor (40 units/μl, Promega).

6. Mix contents and spin the tube briefly in a microcentrifuge.

7. Incubate the tube at 42° C. for 2 minutes in an incubator orthermocycler.

8. Add 1 μl of SuperScript II reverse transcriptase (200 units/μl;Invitrogen).

9. Incubate the tube at 42° C. for 1 hour in an incubator orthermocycler.

10. Add 1 μl of 10 μM first strand cDNA 3′-end extending ACP (rG3-ACP,rG2-ACP, or dG3-ACP).

11. Add 0.3 μl of SuperScript II reverse transcriptase (200 units/μl;Invitrogen).

12. Incubate the tube at 42° C. for 30 minutes in an incubator orthermocycler.

13. Incubate the tube at 70° C. for 15 minutes in an incubator orthermocycler to terminate first-strand synthesis.

14. Place the tube on ice or can be stored at −20° C.

Protocol B: First-Strand Full-Length cDNA Synthesis by CapFinder Method

The following primers are used in the CapFinder method (Clontech):

SMART IV ™ Oligonucleotide (SEQ ID NO: 33)5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACG  GCCr (GGG)-3′; and 5′PCR primer(SEQ ID NO: 34) 5′-AAGCAGTGGTATCAACGCAGAGT-3′, and CDS III/3′PCR primer.

1. Combine the followings in a sterile 0.2 ml microcentrifuge tube: 3 μgof total RNA, 1 μl of 10 μM CDS III/3′ PCR primer (Clontech) and 1 μ.lof 10 μM SMART IV Oligonucleotide (Clontech).

2. Add sterile H2O to a final volume of 5 μl. Mix contents and spin thetube briefly in a microcentrifuge.

3. Incubate the tube at 72° C. for 2 minutes.

4. Cool the tube on ice for 2 minutes. Spin down the contents of thetube briefly in a microcentrifuge.

5. Add the following reagents to the same reaction tube: 10 μl of thetotal volume containing 2 μl of 5× first-strand buffer (Clontech), 1 μlof 20 mM DTT, 1 μl of dNTP (10 mM each dATP, dCTP, dGTP, dTTP) and 1 μlof PowerScript Reverse Transcriptase (Clontech).

6. Mix contents and spin the tube briefly in a microcentrifuge.

7. Incubate the tube at 42° C. for 1 hour.

8. Place the tube on ice or can be stored at −20° C.

B. PCR Amplification

Protocol C: Amplification of Target 5′-End cDNA Fragment Using ACPSystem or Conventional 5′-RACE Method

The present example compares the current CapFinder 5′-RACE technologyand ACP-based 5′ RACE method, wherein the current CapFinder 5′-RACEtechnology could not exclude the high background due to residual amountof the primers during the process. In order to demonstrate if the ACP ofthe present invention can eliminate such background problems arisingfrom primers such as the CapFinder primer, SMART IV Oligonucleotide(Clontech), and cDNA synthesis primer, CDS III/3′ PCR primer (Clontech),used in cDNA synthesis, both the ACP-based 5′-RACE and the CapFinder5′-RACE for the amplification of 5′-ends of mouse JunB and beta-actincDNAs were conducted in the same conditions. The mouse JunB mRNA is arelatively rare transcript in mouse 18.5-day-old placenta RNA, whereasmouse beta-actin is a relatively abundant.

1. Combine the following reagents in a sterile 0.2 ml microcentrifugetube: 50 μl of the total volume containing 1 μl of first-strand cDNAprepared from Protocol A or B, 5 μl of 10×PCR buffer (Promega), 5 μl of25 mM MgCl₂, 5 μl of 2 mM dNTP, 1 μl of 10 μM gene-specific 5′-RACEprimer, 1 μl of 10 μM JYC2 or 5′ PCR primer (Clontech), 0.5 μl of TaqPolymerase (5 units/μl; Promega) and 31.5 μlof sterile dH2O.

2. Mix contents and spin the tube briefly in a microcentrifuge.

3. Conduct PCR reaction under the following conditions: 5 min at 94° C.,followed by 30 cycles of 94° C. for 40 seconds, 58° C. for 40 seconds,and 72° C. for 1 min 30 sec; followed by a 5 min final extension at 72°C.

4. Analyze the amplified products by electrophoresis in a 2% agarose gelfollowed by ethidium bromide staining.

The resulting PCR products can be also detected on a denaturingpolyacrylamide gel by autoradiography or non-radioactive detectionmethods such as silver staining (Gottschlich et al., 1997; Kociok etal., 1998), the use of fluoresenscent-labelled oligonucleotides (Baueret al. 1993; Ito et al. 1994; Luehrsen et al., 1997; Smith et al.,1997), and the use of biotinylated primers (Korn et al., 1992; Tagle etal., 1993; Rosok et al., 1996).

As shown in FIG. 16, the CapFinder methods for mouse JunB and beta-actin5′-RACE using the 5′ PCR primer (Clontech) and the gene-specific primerproduced high backgrounds such as DNA smear (lanes 1 and 3) as describedby many researchers (Chenchik et al., 1998; Matz et al., 1999; Schrammet al., 2000), whereas the ACP-based 5′-RACE of the present inventiongenerated only a single band which corresponds each to the expected size155-bp or 319-bp of mouse JunB (lane 2) or mouse beta-actin (lane 4)5′-end cDNA fragment, respectively. These examples illustrate that theACP can be used to fundamentally eliminate such background problemsarising from contamination of primers used during cDNA synthesis,without the purification step for the removal of primers used in thecDNA synthesis.

FIG. 17 also shows that the ACP of the subject invention permits thenon-specific products not to be formed, which are generated by theCapFinder method (lane 1). The first-strand cDNA was synthesized eitherby CapFinder method (lane 1) or ACP method (lanes 2, 3, and 4) and then,directly used as template in the subsequent PCR amplification for mouseprolactin-like protein PLP-C alpha 5′-RACE. The PLP-C alpha-specific5′-RACE primer is: PLP-C alpha 5′-GAGAGGAT-AGTTTCAGGGAC-3′ (SEQ ID NO:40). The first-strand cDNA 3′-end extending ACPs comprising either threeriboguanines (rG3-ACP; lane 2), three deoxyriboguanines (dG3-ACP; lane4), or a combination of two riboguanines and one deoxyriboguanine(rG2-ACP; lane 3) at the 3′-end generated 5′-end cDNAs so that a singleband which corresponds to the expected size 506-bp of mouse PLP-C alpha5′-end cDNA fragment was produced from the ACP-based PCR for PLP-C alpha5′-RACE.

Protocol D: Amplification of 5′ Enriched cDNA Fragments Using ACP

The first-strand cDNAs are synthesized using Random dN6-ACP in ProtocolA. The PCR amplification was performed by hot start PCR method in whichthe procedure is to set up the complete reactions without the DNApolymerase and incubate the tubes in the thermal cycler to complete theinitial denaturation step at >90° C. Then, while holding the tubes at atemperature above 70° C., the appropriate amount of DNA polymerase canbe pipetted into the reaction.

1. Combine the following reagents in a sterile 0.2 ml microcentrifugetube: 49.5 μl of the total volume containing 1 μl of first-strand cDNAprepared by Random dN6-ACP in Protocol A, 5 μl of 10×PCR buffer(Promega), 5 μl of 25 mM MgCl₂, 5 μl of 2 mM dNTP, 1 μI of 10 μM JYC2(3′ primer), 1 μl of 10 μM JYC4 (5′ primer) and 31.5 μl sterile dH2O.

2. Mix contents and spin the tube briefly in a microcentrifuge.

3. Place the tube in the preheated thermal cycler at 94° C.

4. Add the 0.5 μl of Taq polymerase (5 units/μl; Promega) into thereaction, while holding the tube at the temperature 94° C.

5. Conduct PCR reaction under the following conditions: 5 min at 94° C.followed by 30 cycles of 94° C. for 40 seconds, 68° C. for 40 seconds,and 72° C. for 1 min 30 sec; followed by a 5 min final extension at 72°C.

6. Analyze the amplified products by electrophoresis in a 2% agarose gelfollowed by ethidium bromide staining.

The resulting PCR products can be also detected on a denaturingpolyacrylamide gel by autoradiography or non-radioactive detectionmethods such as silver staining (Gottschlich et al., 1997; Kociok etal., 1998), the use of fluoresenscent-labelled oligonucleotides (Baueret al. 1993; Ito et al. 1994; Luehrsen et al., 1997; Smith et al.,1997), and the use of biotinylated primers (Korn et al., 1992; Tagle etal., 1993; Rosok et al., 1996).

Protocol E: Amplification of Full-Length Enriched cDNAs Using ACP

The first-strand cDNAs are synthesized using Oligo VdT15-ACP in ProtocolA. The PCR amplification was performed by hot start PCR method as inProtocol D.

1. Combine the following reagents in a sterile 0.2 μl microcentrifugetube: 49.5 μl of the total volume containing 1 μl of first-strand cDNAprepared by Oligo VdT15-ACP in Protocol A, 5 μl of 10×PCR buffer(Prornega), 5 μl of 25 mM MgCl₂, 5 μl of 2 mM dNTP, 1 μl of 10 μM JYC2(3′ primer), 1 μl of 10 μM JYC4 (5′ primer) and 31.5 μl of sterile dH2O.

2. Mix contents and spin the tube briefly in a microcentrifuge.

3. Place the tube in the preheated thermal cycler at 94° C.

4. Add the 0.5 μl of Taq polymerase (5 units/μl; Prornega, Madison, USA)into the reaction, while holding the tube at the temperature 94° C.

5. Conduct PCR reaction under the following conditions: 5 min at 94° C.followed by 30 cycles of 94° C. for 40 seconds, 68° C. for 40 seconds,and 72° C. for 1 min 30 sec; followed by a 5 min final extension at 72°C.

6. Analyze the amplified products by electrophoresis in a 2% agarose gelfollowed by ethidium bromide staining.

The resulting PCR products can be also detected on a denaturingpolyacrylamide gel by autoradiography or non-radioactive detectionmethods such as silver staining (Gottschlich et al., 1997; Kociok etal., 1998), the use of fluoresenscent-labelled oligonucleotides (Baueret al. 1993; Ito et al. 1994; Luehrsen et al., 1997; Smith et al.,1997), and the use of biotinylated primers (Korn et al., 1992; Tagle etal., 1993; Rosok et al., 1996).

To evaluate the efficiency of the method using ACP in the amplificationof full-length cDNAs, the full-length cDNAs amplified by either theabove procedures of ACP method or the current CapFinder method wereblotted to a Hybond-N membrane (Amersham/United States Biochemical). Themouse glyceraldehydes-3-phosphate dehydrogenase (GAPDH) cDNA was labeledwith [alpha-32P]dCTP using a random labeling kit (Roche Diagnostics Co,Indianapolis, USA) and used as a probe.

As shown in FIG. 18, the GAPDH cDNA probe detected a single band whichcorresponds to the expected size 1.3-kb of full-length GAPDH cDNA. Asexpected, the signals of the PCR products generated by the above ACPmethod (lane 2) were several fold stronger than the ones by theCapFinder method (lane 1). This example illustrates that the ACP methodof the present invention much more effectively amplifies full-lengthcDNAs than the CapFinder method does.

Example 7 Genomic Fingerprinting Using ACP-Based Arbitrarily Primed PCR

The ACP of the subject invention has been applied to detectpolymorphisms in mouse. The genomic DNAs of mouse strains C57BL/6J, CBA,BALB/cJ, NOR, SPRETUS, PANCEVO, and Korean Wild Mouse were used startingmaterials. Genomic DNA was prepared from the liver of mice using theQIAamp Tissue Kit (QIAGEN, Hilden, Germany). The arbitrary ACPs used inthe subject invention are:

ACP101 (SEQ ID NO. 64) 5′-GTCTACCAGGCATTCGCTTCATIIIIICCGGAGGATC-3′;ACP109 (SEQ ID NO. 65) 5′-GTCTACCAGGCATTCGCTTCATIIIIICTGCAGGACG-3 ′; andACP116 (SEQ ID NO. 66) 5′-GTCTACCAGGCATTCGCTTCATIIIIICGGAGCATCC-3′.

A set of arbitrary ACPs, ACP101 and ACP109 (FIG. 19A), or ACP101 and ACP116 (FIG. 19B), was used as primers for mouse genomic fingerprinting.The PCR amplification was performed by hot start PCR method as describedin Example 2. The genomic fingerprinting using ACP is conducted by twostages of PCR amplifications under the following conditions:amplification reactions are performed under low stringent conditions bytwo cycles of the first-stage PCR comprising annealing, extending anddenaturing reaction; the reaction mixture in the final volume of 49.5 μlcontaining 50 ng of the genomic DNA, 5 μl of 10×PCR reaction buffer(Promega), 5 μl of 25 mM MgC 12, 5 μl of dNTP (2 mM each dATP, dCTP,dGTP, dTTP), each 7 μl of a pair of ACPs (each 10 μM) is pre-heated at94° C., while holding the tube containing the reaction mixture at the94° C., 0.5 μl of Taq polymerase (5 units/μl; Promega) is added into thereaction mixture; the PCR reactions are as follows: two cycles of 94° C.for 40 sec, 52° C. for 3 min, and 72° C. for 1 min; followed bydenaturing the amplification product at 94° C.; after the completereaction of the first-stage PCR, 4 μl of the pre-selected arbitraryprimer JYC4 (10 μM) corresponding to the 5′-end portion of the ACPs areadded to the reaction mixture and then the second stage PCRamplification is conducted as follows: 40 cycles of 94° C. for 40 sec,68° C. for 40 sec, and 72° C. for 40 sec; followed by a 5 min finalextension at 72° C.

Amplification products were resolved and analyzed by electrophoresis ina 2.0 agarose gel which was stained with ethidium bromide andphotographed. The resulting PCR products can be also detected on adenaturing polyacrylamide gel by autoradiography or non-radioactivedetection methods such as silver staining (Gottschlich et al., 1997;Kociok et al., 1998), the use of fluoresenscent-labelledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), and the use of biotinylated primers (Korn etal., 1992; Tagle et al., 1993; Rosok et al., 1996).

FIG. 19 shows the results of an experiment in which a pair of arbitraryACPs were used to amplify segments of genomic DNA from a variety ofmouse strains. To examine the reproducibility of genomic fingerprinting,the fingerprinting of each mouse strain was duplicated using twodifferent sets of ACPs. The ACP-based PCR amplification produced severalDNA segments from each set of primers and the results were reproducible.The polymorphisms were apparent between mice stains, indicating thatmice stains can be distinguished through polymorphisms in genomicfingerprintings generated by ACP-based arbitrarily primed PCR. Thus, theACP of the subject invention is useful to detect polymorphisms andconstruct genetic maps.

Example 8 Multiplex PCR Using ACP-Based PCR

To demonstrate the application of ACP in multiplex PCR, the portionscontaining single nucleotide polymorphisms of human leukocyte adhesionmolecule I (ELAM1) and human p53 (TP53) genes were amplified with eitherconventional primers or ACP. The process and results for the multiplexPCR amplification using ACPs are described herein. DNA template wasobtained from human placenta.

The conventional primers for exon3 of ELAM1 (155 bp) used in the Exampleare:

ELAM1N1 (SEQ ID NO: 67) 5′-TTGCACACTGTTGATTCTAA-3′; and ELAM1C1(SEQ ID NO: 68) 5′-TTATTGATGGTCTCTACACA-3′.

The conventional primers for exon10 of ELAMI (287 bp) used in theExample are:

ELAMIN2 (SEQ ID NO: 69) 5′-CCACTGAGTCCAACATTC-3′;; and ELAM1C2(SEQ ID NO: 70) 5′-CTGAAACACTTCCCACAC-3′.

The conventional primers for exon4 of TP53 (349 bp) used in the Exampleare:

P53N1 (SEQ ID NO: 71) 5′-CCTCTGACTGCTCTTTTCAC-3′; And P53C1(SEQ ID NO: 72) 5′-ATTGAAGTCTCATGGAAGCC-3′.

The conventional primers for exons7-8 of TP53 (750 bp) used in theExample are:

P53N2 (SEQ ID NO: 73) 5′-TGCTTGCCACAGGTCTC-3′; and P53C2 (SEQ ID NO: 74)5′-GCAGTGCTAGGAAAGAGG-3′.

These conventional primers used in the Example are known as the primersthat generate non-specific products in conventional mutiplex PCR methodsas known in the art.

The ACPs of the subject invention were applied to these fourconventional primer sets to demonstrate if the ACP can overcome theproblems such as non-specific products resulting from the use of theseconventional primer sets for multiplex PCR.

The 3′-end portions of the ACPs comprise the sequences of the aboveconventional primers as follows and thus the size of ACPs is 26 by or 27by bigger than that of the conventional primers:

ELAM1N1-ACP (SEQ ID NO: 75)5′-GTCTACCAGGCATTCGCTTCATIIIIITTGCACACTGTTGATTCTA A-3′; ELAM1C1-ACP(SEQ ID NO: 76) 5′-TCACAGAAGTATGCCAAGCGAIIIIITTATTGATGGTCTCTACAC A-3′;ELAMIN2-ACP (SEQ ID NO: 77)5′-GTCTACCAGGCATTCGCTTCATIIIIICCACTGAGTCCAACATT C-3′; ELAM1C2-ACP(SEQ ID NO: 78) 5′-TCACAGAAGTATGCCAAGCGAIIIIICTGAAACACTTCCCACAC-3;P53N1-ACP (SEQ ID NO: 79)5′-GTCTACCAGGCATTCGCTTCATIIIIICCTCTGACTGCTCTTTTC AC-3′; P53 C1-ACP(SEQ ID NO: 80) 5′-TCACAGAAGTATGCCAAGCGAIIIIIATTGAAGTCTCATGGAAGC C3′;P53N2-ACP (SEQ ID NO: 81)5′-GTCTACCAGGCATTCGCTTCATIIIIITGCTTGCCACAGGTCTC- 3′; and P53C2-ACP(SEQ ID NO: 82) 5′-TCACAGAAGTATGCCAAGCGAIIIIIGCAGTGCTAGGAAAGAGG- 3′.

The 5′-end portion sequences of the ACPs comprise and serve aspre-selected arbitrary primer sequences only for the second-stage PCRamplification:

(SEQ ID NO: 11) JYC3 5′-TCACAGAAGTATGCCAAGCGA-3′ and (SEQ ID NO: 12)JYC4 5′-GTCTACCAGGCATTCGCTTCAT-3.′

Multiplex PCR amplifications were conducted by one-stop or non-stoptwo-stage PCR amplifications, which is a unique feature of the presentinvention. The PCR amplification was performed by hot start PCR methodas described in Example 2.

Protocol A: One-Stop Two-Stage PCR Amplifications

(A) First-Stage PCR Amplification

The first-stage PCR amplification was conducted by two cycles of PCRcomprising of annealing, extending and denaturing reaction; the reactionmixture in the final volume of 49.5 containing 50 ng of human genomicDNA, 8 μl of 10×PCR reaction buffer (Promega), 7 μl of 25 mM MgCl₂, 5μla of dNTP (2 mM each dATP, dCTP, dGTP, dTTP), each 0.5 μl of each 5′ACP (10 μM) and 3′ ACP (10 μM) set is pre-heated at 94° C., whileholding the tube containing the reaction mixture at the 94° C., 0.5 μlof Taq polymerase (5 units/μl; Promega) is added into the reactionmixture; the PCR reactions are as follows: two cycles of 94° C. for 40sec, 60° C. for 40 sec, and 72° C. for 40 sec; followed by denaturingthe amplification product at 94° C.

(B) Second-Stage PCR Amplification

The resultant products generated by the first-stage PCR amplificationusing multiple sets of the ACPs were then amplified by the followingsecond-stage PCR amplification under higher annealing temperature. Afterthe completion of the first-stage PCR amplification, each 2 μl of 10 μMpre-selected arbitrary primers, JYC3 and JYC4, was added into thereaction mixture obtained from the first-stage PCR amplification, underdenaturing temperature such as at 94° C. The second stage-PCR reactionwas as follows: 40 cycles of 94° C. for 40 sec, 68° C. for 40 sec, and72° C. for 1 min; followed by a 5 min final extension at 72° C.

The amplified products were analyzed by electrophoresis in a 2% agarosegel and detected by staining with ethidium bromide. The resulting PCRproducts can be also detected on a denaturing polyacrylamide gel byautoradiography or non-radioactive detection methods such as silverstaining (Gottschlich et al., 1997; Kociok et al., 1998), the use offluoresenscent-labelled oligonucleotides (Bauer et al. 1993; Ito et al.1994; Luehrsen et al., 1997; Smith et al., 1997), and the use ofbiotinylated primers (Korn et al., 1992; Tagle et al., 1993; Rosok etal., 1996).

FIGS. 20 and 21 show the results of experiments in which three or foursets of primers were used to amplify mutiplex segments of genomic DNA atone reaction. The conventional primer sets generated non-specificproducts as well as specific-target products from three sets (FIG. 20A)or four sets (FIG. 21A) of primers. In contrast, the three sets (FIG.20B) or four sets (FIG. 21 B) of ACPs produced only mutiplex targetproducts. Thus, the ACP of the subject invention can be used for theapplication of multiplex PCR.

Protocol B: Non-Stop Two-Stage PCR Amplifications

Alternatively, the complete sequences of each ACP set, instead of thepre-selected arbitrary primers such as JYC3 and JYC4, can be used asprimers for the second-stage PCR amplification at the high stringentconditions. In this case, it is not necessary to add the pre-selectedarbitrary primers to the reaction mixture at the time of or after thefirst-stage PCR reaction.

The process of the non-stop two-stage PCR amplifications is basicallyidentical to Protocol A, except that the second stage PCR amplificationshould immediately follow first stage PCR amplification without anydelay because there is no step of adding pre-selected arbitrary primersand that the concentration of each ACP set, each 1 μl of 5′ ACP (10 μM)and 3′ ACP (10 μM) set, is added at the first stage PCR amplification.

The amplified products were analyzed by electrophoresis in a 2% agarosegel and detected by staining with ethidium bromide. The resulting PCRproducts can be also detected on a denaturing polyacrylamide gel byautoradiography or non-radioactive detection methods such as silverstaining (Gottschlich et al., 1997; Kociok et al., 1998), the use offluoresenscent-labelled oligonucleotides (Bauer et al. 1993; Ito et al.1994; Luehrsen et al., 1997; Smith et al., 1997), and the use ofbiotinylated primers (Korn et al., 1992; Tagle et al., 1993; Rosok etal., 1996).

Consistent with the results of one-stop two-stage PCR amplifications(FIG. 21B), non-stop two-stage PCR amplification also produced onlytarget multiplex specific products (FIG. 21 C). These examplesillustrate that ACP permits the products to be free from the backgroundproblems as well as non-specificity arising from the conventionalprimers used in multiplex PCR methods as known in the art.

Example 9 Identification of Conserved Homology Segments in MultigeneFamilies Using ACP

The ACP of the subject invention was applied to detect and cloneconserved homology segments in multigene families. In the presentexample, degenerate primers were designed to detect homeobox sequences.The homeobox genes are characterized by a conserved 180-bp nucleotidesequence known as the homeobox, which encodes a 60-aa DNA bindinghomeodomain. To isolate homeobox genes involved in mouse embryodevelopment, total RNA obtained from three different stages of conceptusdevelopment, mouse 4.5-, 11.5-, and 18.5-day-old conceptuses, was usedas a starting material. First-strand cDNAs were prepared under the sameconditions as used in the cDNA synthesis of Example 3, whereinJYC5-T15-ACP was used as the first-strand cDNA synthesis primer.

The following ACPs comprise the degenerate sequences for homeoboxsequence at their 3′-end portions and were used as degeneratehomeobox-specific primers for the first-stage PCR amplification:

JYC2-HD1 (SEQ ID NO: 83) 5′-GCTTGACTACGATACTGTGCGAIIIIIGTNCRRGTGTGGTT-3;JYC2-HD2 (SEQ ID NO: 84)5′-GCTTGACTACGATACTGTGCGAIIIIIGTNCRRGTCTGGTT-3′; and JYC2-ED3(SEQ ID NO: 85) 5′-GCTTGACTACGATACTGTGCGAIIIIIGTNCRRGTTTGGTT-3′.

The PCR amplification was performed by hot start PCR method as describedin Example 2 and conducted by one-stop or non-stop two-stage PCRamplifications.

The following is an example of the process of one-stop two-stage PCRamplifications.

1. Combine the following reagents in a sterile 0.2 ml microcentrifugetube: 49.5 μl of the total volume containing 1 μl of first-strand cDNA(50 ng/μl), 5 μl of 10×PCR buffer (Roche), 5 μl of 2 mM dNTP, 1 μl ofone of 10 μM JYC2HD1, JYC2-HD2, or JYC2-HD3 (5′ primer), 1 μl of 10 μMJYC5-T15-ACP (3′ primer) and 36.5 μl of sterile dH2O.

2. Mix contents and spin the tube briefly in a microcentrifuge.

3. Place the tube in the preheated thermal cycler at 94° C.

4. Add 0.5 μl of Taq polymerise (5 units/μl; Roche) into the reactionwhile holding the tube at the temperature 94° C.

5. Conduct PCR reaction under the following conditions: one cycle of 94°C. for 1 min, 52° C. for 3 min, and 72° C. for 1 min; followed by 40cycles of 94° C. for 40 sec, 65° C. for 40 sec, and 72° C. for 40 sec;and followed by a 5 min final extension at 72° C.

The amplified products were analyzed by electrophoresis in a 2% agarosegel and detected by staining with ethidium bromide. The resulting PCRproducts can be also detected on a denaturing polyacrylamide gel byautoradiography or non-radioactive detection methods such as silverstaining (Gottschlich et al., 1997; Kociok et al., 1998), the use offluoresenscent-labelled oligonucleotides (Bauer et al. 1993; Ito et al.1994; Luehrsen et al., 1997; Smith et al., 1997), and the use ofbiotinylated primers (Korn et al., 1992; Tagle et al., 1993; Rosok etal., 1996).

Many differentially expressed bands in a specific stage were obtained,subcloned into the pGEM-T Easy vector (Promega), and sequenced. Sequenceanalysis reveals that some of the clones contain homeobox sequences.Northern blot or RT-PCR analysis shows that the clones are identical tothe results of the expression patterns observed by the electrophoresis.These results indicate that the method using the ACP of the presentinvention for isolating conserved homology segments in multigenefamilies produces only real PCR products. Freedom from false positives,which is one major bottleneck remaining in the previous PCR-basedtechniques for isolating conserved homology segments in multigenefamilies, allows avoiding the subsequent labor-intensive work requiredfor the verification of the amplified cDNA fragments.

Example 10 Single Nucleotide Polymorphism Genotyping Using ACP-Based PCR

To demonstrate the application of ACP in single nucleotide polymorphismgenotyping, a portion containing a single nucleotide polymorphism (SNP)of human p53 (TP53) gene was amplified with either conventional primeror ACP. The process and results for the SNP genotyping using ACPs aredescribed herein. DNA templates were obtained from human blood sampleswhich have a SNP in exon 4 of the TP53 gene. This polymorphism isexpressed as an Arg→Pro substitution at amino acid position 72 byreplacing G with C. A 349 nt sequence between nucleotide 11991 and 12339of the TP53 gene was amplified from each type of template by a set ofthe following primers:

P53N (SEQ ID NO: 86) 5′-CCTCTGACTGCTCTTTTCAC-3′ and P53C-ACP(SEQ ID NO: 87) 5′-TCACAGAAGTATGCCAAGCGAIIIIIATTGAAGTCTCATGGAAGC C-3′.

The amplified products containing the SNP between their ends were usedas templates for detecting the SNP using allele-specific ACPs asfollows:

P53N1A-ACP (SEQ ID NO: 88) 5′-GTCTACCAGGCATTCGCTTCATIIIIICCCC GCGTGG-3′, P53N1B-ACP (SEQ ID NO: 89) 5′-GTCTACCAGGCATTCGCTTCATIIIIICCCCC CGTGG-3′, P53N2A-ACP (SEQ ID NO: 90)5′-GTCTACCAGGCATTCGCTTCATIIIIICCCC C CGTGG-3′, P53N2B-ACP(SEQ ID NO: 91) 5′-GTCTACCAGGCATTCGCTTCATIIIIITCCCC C CGTG-3′,P53N3A-ACP (SEQ ID NO: 92) 5′-GTCTACCAGGCATTCGCTTCATIIIIICTCCCC GCGT-3′, P53N3B-ACP (SEQ ID NO: 93) 5′-GTCTACCAGGCATTCGCTTCATIIIIICTCCCCC CGT-3′, P53N4A-ACP (SEQ ID NO: 94)5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC G CG-3′, P53N4B-ACP(SEQ ID NO: 95) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC C CG-3′,P53N5A-ACP (SEQ ID NO: 96) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC G -3′,and P53N5B-ACP (SEQ ID NO: 97) 5′-GTCTACCAGGCATTCGCTTCAIIIIIIGCTCCCC C-3′.

The polymorphic base is underlined at the 3′-end portion of eachallele-specific ACP and the position of the polymorphic base isconsidered an interrogation position. The interrogation position isplaced at several different positions from the 3′-end of allele-specificACPs in order to determine the most critical position in annealingspecificity for detecting the SNP.

The allele-specific ACPs were used as 5′ primers. P53C-ACP and one ofP53N1A-ACP, P53N2A-ACP, P53N3A-ACP, P53N4A-ACP and P53N5A-ACP were usedfor wild-type A genotyping. P53C-ACP and one of P53N1B-ACP, P53N2B-ACP,P53N3BACP, P53N4B-ACP and P53N5B-ACP were used for variant-type Bgenotyping. The 5′-end portion sequences of the ACPs were served aspre-selected arbitrary primer sequences for the second-stage PCRamplification:

(SEQ ID NO: 11) JYC3 5′-TCACAGAAGTATGCCAAGCGA-3′ and (SEQ ID NO: 12)JYC4 5′-GTCTACCAGGCATTCGCTTCAT-3′.

(A) First-Stage PCR Amplification

The first-stage PCR amplification was conducted by one cycle of PCRconsisting of annealing, extending and denaturing reaction; the reactionmixture in a final volume of 49.5 μl containing 1 μl of the amplifiedtarget genomic segment containing the SNP in exon 4 of the TP53 gene, 5μl of 10×PCR reaction buffer (Promega), 5 μl of 25 mM MgCl₂, 5 μl ofdNTP (2 mM each dATP, dCTP, dGTP, dTTP), and 1 μl of one ofallele-specific ACPs (10 μM) is pre-heated at 94° C., while holding thetube containing the reaction mixture at the 94° C., 0.5 μl of Taqpolymerase (5 units/μl; Promega) is added into the reaction mixture; forthe allele-specific ACPs having 10 nucleotides at its 3′-end portion,the PCR reactions are as follows: one cycle of 94° C. for 40 sec, 55° C.for 40 sec, and 72° C. for 40 sec; followed by denaturing theamplification product at 94° C.; for the allele-specific ACPs having 8nucleotides at its 3′-end portion, the PCR reactions are as follows: onecycle of 94° C. for 40 sec, 50° C. for 40 sec, and 72° C. for 40 sec;followed by denaturing the amplification product at 94° C. The resultantproduct is a first DNA strand complementary to the target genomicsegment.

(B) Second-Stage PCR Amplification

The resultant product generated by the first-stage PCR amplification wasthen amplified by the following second-stage PCR amplification at ahigher annealing temperature than the first annealing temperature. Afterthe completion of the first-stage PCR amplification, 1 μl of 10 μMpre-selected arbitrary primer, JYC3, was added into the reaction mixtureobtained from the first-stage PCR amplification, under denaturingtemperature such as at 94° C. The second stage-PCR reaction wasperformed as follows: 30 cycles of 94° C. for 40 sec, 68° C. for 40 sec,and 72° C. for 40 sec; followed by a 5 min final extension at 72° C.

The amplified products were analyzed by electrophoresis in a 2% agarosegel and detected by staining with ethidium bromide. The resulting PCRproducts can be also detected on a denaturing polyacrylamide gel byautoradiography or non-radioactive detection methods such as silverstaining (Gottschlich et al., 1997; Kociok et al., 1998), the use offluoresenscent-labelled oligonucleotides (Bauer et al. 1993; Ito et al.1994; Luehrsen et al., 1997; Smith et al., 1997), and the use ofbiotinylated primers (Korn et al., 1992; Tagle et al., 1993; Rosok etal., 1996).

FIG. 22 shows the results of allele-specific amplification using ACP.The pair of wild-type A-specific ACPs (P53N2A-ACP and P53C-ACP)generated a specific target product only from the samples havinghomozygous wild-type A (lane 1) or heterozygous genotyping (lane 3), butnot from the samples having homozygous variant-type B genotyping (lane5). The pair of variant-type B-specific ACPs (P53N2B-ACP and P53CACP)generated a specific target product only from the samples havinghomozygous variant-type B (lane 6) or heterozygous genotyping (lane 4),but not from the samples having homozygous wild-type A genotyping (lane2). These results indicate that the ACP of the subject invention can beapplied as an easy and economic method for detecting the genotype ofSNPs since the use of fluorescent DNA probe nor post-PCR processing isnot required in this approach. The allele-specific ACPs each having aninterrogation position at its 3′-end portion showed improvement ofannealing specificity. Moreover, when the allele-specific ACP has aninterrogation position at the position 5 from the 3′-end (e.g.,P53N2AACP and P53N2B-ACP), the annealing specificity is most criticallyaccomplished.

In order to verify if the position 5 from the actual 3′-end of theallele-specific ACP is the most appropriate for the interrogationposition, additional six experiments were conducted using the sameprocess as used in FIG. 22. DNA templates were obtained from human bloodsamples which have a SNP. Six short genomic fragments containing SNPswere amplified using each different primer set as follows:

703N (SEQ ID NO: 98) 5′-ATTCTGATGGTGTGGATTGTG-3′ and SM703C(SEQ ID NO: 99) 5′-TCACAGAAGTATGCCAAGCGAIIIIIACCCTGGAGTAGACGAAG A-3′for Beta-2 adrenergic receptor (ADRB2), 028N (SEQ ID NO: 102)5′-CCTTCTGTGCTTGATGCTTTT-3′ and SMO28C (SEQ ID NO: 103)5′-TCACAGAAGTATGCCAAGCGAIIIIICAGGAAGGATGAGCATTTA G-3′for Chemokine (c-c motif) receptor 5 (CCR5), 695N: (SEQ ID NO: 106)5′-AGAAAAACCAGAGGCAGCTT-3′ and SM695C (SEQ ID NO: 107)5′-TCACAGAAGTATGCCAAGCGAIIIIIAGCACAAACCAAAGACACAG T-3′for Interleukin 13 receptor, 679N (SEQ ID NO: 110)5′-CTAGCTGCAAGTGACATCTCT-3′ and SM679C (SEQ ID NO: 111)5′-TCACAGAGTATCCAAGCGIIIIITCAGTAAGAAGCCAGGAGAG-3′for Leukocyte adhesion molecule-1 (LAM-1), 832N (SEQ ID NO: 114)5′-TTTTGGGTGGAGGCTAACAT-3′ and SM832C: (SEQ ID NO: 115)5′-TCACAGAAGTATGCCAGCGAIIIIIAACGATGCAGACACCACCA-3′for Tachykinin receptor 3 (TACR3), and 880N (SEQ ID NO: 118)5′-CTTCCACCAATACTCTTTTCC-3′ and SM880C: (SEQ ID NO: 119)5′-TCACAGAAGTATGCCAGCGAIIIII GCATACACACAAGAGGCAG A-3′for Interleukin 1, beta (IL1B).

The amplified products containing the SNP between their ends were usedas templates for detecting the SNPs wherein allele-specific ACPs wereapplied as follows:

SM703-A (SEQ ID NO: 100) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGGTAC A GGGC-3′and SM703-B (SEQ ID NO: 101) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGGTAC CGGGC-3′ for Beta-2 adrenergic receptor (ADRB2), SM028-A (SEQ ID NO: 104)5′-GTCTACCAGGCATTCGCTTCATIIIIITCCAA A CCAA-3′ and SM028-B(SEQ ID NO: 105) 5′-GTCTACCAGGCATTCGCTTCATIIIIITCCAA C CCAA-3′for Chemokine (c-c motif) receptor 5 (CCR5), SM695-A (SEQ ID NO: 108)5′-GTCTACCAGGCATTCGCTTCATIIIII CCATT T TAGG-3′ and SM695-B(SEQ ID NO: 109) 5′-GTCTACCAGGCATTCGCTTCATIIIII CCATT G TAGG-3′for Interleukin 13 receptor, SM679-A (SEQ ID NO: 112)5′-GTCTACCAGGCATTCGCTTCATIIIIICCAGA A CTTT-3′ and SM679-B(SEQ ID NO: 113) 5′-GTCTACCAGGCATTCGCTTCATIIIIICCAGA C CTTT-3′for Leukocyte adhesion molecule-1(LAM-1), SM832-A (SEQ ID NO: 116)5′-GTCTACCAGGCATTCGCTTCATIIIIIGACTG G TAAA-3′ and SM832-B(SEQ ID NO: 117) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGACTG A TAAA-3′for Tachykinin receptor 3 (TACR3), and SM880-A (SEQ ID NO: 120)5′-GTCTACCAGGCATTCGCTTCATIIIIIAAAGC C ATAA-3′ and SM880-B(SEQ ID NO: 121) 5′-GTCTACCAGGCATTCGCTTCATIIIIIAAAGC T ATAA-3′for Interleukin 1, beta (IL1B).

FIG. 23 shows the results of allele-specific amplifications for sixadditional SNPs each present in different gene such as Beta-2 adrenergicreceptor (ADRB2) (A), Chemokine (c-c motif) receptor 5 (CCR5) (B),Interleukin 13 receptor (C), Leukocyte adhesion molecule-1 (LAM-1) (D),Tachykinin receptor 3 (TACR3) (E), and Interleukin 1, beta (IL1B) (F).Consistant with the results of FIG. 22, the annealing specificity iscritically accomplished when the allele-specific ACP has aninterrogation position at the position 5 from the 3′-end. The pair ofwild-type A-specific ACPs generated a specific target product only fromthe samples having homozygous wild-type A (lane 1) or heterozygousgenotyping (lane 3), but not from the samples having homozygousvariant-type B genotyping (lane 5). The pair of variant-type B-specificACPs generated a specific target product only from the samples havinghomozygous variant-type B (lane 6) or heterozygous genotyping (lane 4),but not from the samples having homozygous wild-type A genotyping (lane2).

Having described a preferred embodiment of the present invention, it isto be understood that variants and modifications thereof falling withinthe spirit of the invention may become apparent to those skilled in thisart, and the scope of this invention is to be determined by appendedclaims and their equivalents.

TABLE 1 SEQ ID NO Designation Sequence Information 1 ACP 15′-GTCTACCAGGCATTCGCTTCATIIIIICAGGAGTGG-3′ 2 ACP25′-GTCTACCAGGCATTCGCTTCATIIIIIGGCGACGATS-3′ 3 ACP35′-GTCTACCAGGCATTCGCTTCATIIIIIGCCATCGACS-3′ 4 ACP45′-GTCTACCAGGCATTCGCTTCATIIIIIAGATGCCCGW-3′ 5 ACP55′-GTCTACCAGGCATTCGCTTCATIIIIIAGGCGATGCS-3′ 6 ACP65′-GTCTACCAGGCATTCGCTTCATIIIIITCTCCCGGTS-3′ 7 ACP75′-GTCTACCAGGCATTCGCTTCATIIIIITTGTGGCGGS-3′ 8 ACP85′-GTCTACCAGGCATTCGCTTCATIIIIICTCCGATGCS-3′ 9 ACP95′-GTCTACCAGGCATTCGCTTCATIIIIICCTGCGGGTW-3′ 10 JYC25′-GCTTGACTACGATACTGTGCGA-3′ 11 JYC3 5′-TCACAGAAGTATGCCAAGCGA-3′ 12 JYC45′-GTCTACCAGGCATTCGCTTCAT-3′ 13 ACP105′-GTCTACCAGGCATTCGCTTCATIIIIIGCCATCGACC-3′ 14 ACP115′-GTCTACCAGGCATTCGCTTCATIIIIIGCCATCGACG-3′ 15 ACP125′-GTCTACCAGGCATTCGCTTCATIIIIIAGGCGATGCC-3′ 16 ACP135′-GTCTACCAGGCATTCGCTTCATIIIIIAGGCGATGCG-3′ 17 ACP145′-GTCTACCAGGCATTCGCTTCATIIIIICTCCGATGCC-3′ 18 ACP155′-GTCTACCAGGCATTCGCTTCATIIIIICTCCGATGCG-3′ 19 CRP2105′-GTCTACCAGGCATTCGCTTCATGCCATCGACC-3′ 20 ACP165′-GTCTACCAGGCATTCGCTTCATIIGCCATCGACC-3′ 21 ACP175′-GTCTACCAGGCATTCGCTTCATIIIIGCCATCGACC-3′ 22 ACP185′-GTCTACCAGGCATTCGCTTCATIIIIIIGCCATCGACC-3′ 23 ACP195′-GTCTACCAGGCATTCGCTTCATIIIIIIIIGCCATCGACC-3′ 24 dT-JYC35′-CACAGAAGTATGCCAAGCGACTCGAGTTTTTTTTTTTTTTT-3′ 25 dT-JYC25′-GCTTGACTACGATACTGTGCGATTTTTTTTTTTTTTT-3′ 26 JYC2-T13C5′-CTTGACTACGATACTGTGCGATTTTTTTTTTTTTC-3′ 27 JYC2-T13G5′-GCTTGACTACGATACTGTGCGATTTTTTTTTTTTTG-3′ 28 JYC2-T13A5′-GCTTGACTACGATACTGTGCGATTTTTTTTTTTTTA-3′ 29 dT10-JYC25′-GCTTGACTACGATACTGTGCGATTTTTTTTTT-3′ 30 dT10-ACP15′-GCTTGACTACGATACTGTGGAIIIIITTTTTTTTTT-3′ 31 DEG 2GCCATCGACCCGTTTCTCTAGCCCCATCTTCATGTGTTTTAATGAGATGATATTAATTCA′TTACATTCATGGATAATATGTCCCTGAGTACATTCTAATCTAGATTTAACT TCAAAAAAAAAAAAAAAAA 32 DEG 5AGGCGATGCGGGCTGTACTCTGGGTGGCTGCCACAGTCTCATGAGAAACCAAGGGCAAAGGACCAAGGAAAAGGGTCTCAGGCCCCTAAAGCAGTGGCTTTCAACCATCCTAATGTTGTGACCTTTTAATACAGTTCCTCATGTTGTGTGACCCCCCAACCATAAAATGA′TTTTTGTTTCTACTTC AAAAAAAAAAAAAAAAAAAAAA 33SMART IV 5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACG GCCr(GGG)-3′ 34 5′ PCR5′-AAGCAGTGGTATCAACGCAGAGT-3 Primer 35 CDS 111/3′5′-ATTCTAGAGGCCGAGGCGGCCGACATG-(dT)₃₀ VN-3′ 36 rG3-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIIGGr(GGG)-3′ 37 rG2-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIIGGr(GG) d (G)-3′ 38 dG3-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIIGGd (GGG)-3′ 39 Oligo dT18-5′-GCTTGACTACGATACTGTGCGAIIIIITTTTTTTTTTTTTTTTTT-3′ 40 PLP-Cα5′-GAGAGGATAGTTTCAGGGAC-3′ 41 JunB3 5′-CTCCGTGGTACGCCTGC′TTTCTC-3′ 42β-actin -1 5′-TCGTCACCCACATAGGAGTC-3′ 43 β-actin -25′-CTAAGAGGAGGATGGTCGC-3′ 44 EsxN7 5′-GCCGGTTGCAGAGCACC-3′ 45 EsxC65′-GAACCATGTTTCTGAATGCC-3′ 46 EsxN7-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIIGCCGGT TGCAGAGCACC-3′ 47 EsxC6-ACP5′-GCTTGACTACGATACTGTGCGAIIIIIGAACCAT GTTTCTGAATGCC-3′ 48 EsxN15′-GAATCTGAAACAACTTTCTA-3′ 49 EsxC2 5′-GATGCATGGGACGAGGCACC-3′ 50EsxN1-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIIGAATCT GAAACAACTTTCTA-3′ 51EsxN3 5′-CGCCGCACCCCTGCCCGCA-3′ 52 EsxC5 5′-GATGCATGGGACGAGGCA-3′ 53EsxN3-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIICGCCGC ACCCCTGCCCGCA-3′ 54Oligo-dT15 5′-TTTTTTTTTTTTTTT-3′ 55 EsxC2-ACP5′-GCTTGACTACGATACTGTGCGAIIIIIGATGCA TGGGACGAGGCACC-3′ 56 EsxC5-ACP5′-GCTTGACTACGATACTGTGCGAIIIIIGATGCA TGGGACGAGGCA-3′ 57 OligoVdT15-5′-GC TTGACTACGATACTGTGCGAIIIIITTTTTT ACP TTTTTTTTTV-3′ 58 dN6-ACP5′-GCTTGACTACGATACTGTGCGAI I IIINNNNNN-3′ 59 rG1-ACP5′GTCTACCAGGCATTCGCTTCATIIIIIGGr(G)d(GG)-3′ 60 JYC55′-CTGTGAATGCTGCGACTACGAT-3′ 61 JYC5-T₁₅-5′-CTGTGAATGCTGCGACTACGATIIIIITTTTTT ACP TTTTTTTTT-3′ 62 JYC5-T₁₅V-5′-CTGTGAATGCTGCGACTACGATIIIIITTTTTTT ACP TTTTTTTTV-3′ 63 JYC5-5′-CTGTGAATGCTGCGACTACGATIIIIITTTTTT T₁₅VN- TTTTTTTTTVN-3′ ACP 64ACP 101 5′-GTCTACCAGGCATTCGCTTCATIIIIICCGGAGGATC-3′ 65 ACP 1095′-GTCTACCAGGCATTCGCTTCATIIIIICTGCAGGACG-3′ 66 ACP 1165′-GTCTACCAGGCATTCGCTTCATIIIIICGGAGCATCC-3′ 67 ELAM1N15′-TTGCACACTGTTGATTCTAA-3′ 68 ELAM1C1 5′-TTATTGATGGTCTCTACACA-3′ 69ELAM1N2 5′-CCACTGAGTCCAACATTC-3′ 70 ELAM1C2 5′-CTGAAACACTTCCCACAC-3′ 71P53N1 5′-CCTCTGACTGCTCTTTTCAC-3′ 72 P53C1 5′-ATTGAAGTCTCATGGAAGCC-3′ 73P53N2 5′-TGCTTGCCACAGGTCTC-3′ 74 P53C2 5′-GCAGTGCTAGGAAAGAGG-3′ 75ELAM1N1- 5′-GTCTACCAGGCATTCGCTTCATIIIIITTGCACACTGT ACP TGATTCTAA-3′ 76ELAM1C1- 5′-TCACAGA A GTATGCCAAGCGAIIIIITTATTGATGGT ACP CTCTACACA-3′ 77ELAM1N2- 5′-GTCTACCAGGCATTCGCTTCATIIIIICCACTGAGTCC ACP AACATTC-3′ 78ELAM1C2- 5′-TCACAGAAGTATGCCAAGCGAIIIIICTGAAACACTT ACP CCCACAC-3′ 79P53N1-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIICCTCTGACTGC TCTTTTCAC-3′ 80P53C1-ACP 5′-TCA CAGAAGTATGCCAAGCGAIIIIIATTGAAGTCTC ATGGAAGCC-3′ 81P53N2-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIITGCTTGCCACA GGTCTC-3′ 82P53C2-ACP 5′-TCACAGAAGTATGCCAAGCGAIIIIIGCAGTGCTAGG AAAGAGG-3′ 83JYC2-HD1 5′-GCTTGACTACGATACTGTGCGAIIIIIGTNCRRGTGTG GTT-3′ 84 JYC2-HD25′-GCTTGACTACGATACTGTGCGAIIIIIGTNCRRGTCTG GTT-3′ 85 JYC2-HD35′-GCTTGACTACGATACTGTGCGAIIIIIGTNCRRGTTTG GTT-3′ 86 P53N5′-CCTCTGACTGCTCTTTTCAC-3′ 87 P53C-ACP5′-TCACAGAAGTATGCCAAGCGAIIIIIATTGAAGTCTC ATGGAAGCC-3′ 88 P53N1A-5′-GTCTACCAGGCATTCGCTTCATIIIIICCCC G CGTGG-3′ ACP 89 P53N1B-5′-GTCTACCAGGCATTCGCTTCATIIIIICCC C CCGTGG-3′ ACP 90 P53N2A-5′-GTCTACCAGGCATTCGCTTCATIIIIITCCCC G CGTG-3′ ACP 91 P53N2B-5′-GTCTACCAGGCATTCGCTTCATIIIIITCCCC C CGTG-3′ ACP 92 P53N3A-5′-GTCTACCAGGCATTCGCTTCATIIIIICTCCCC G CGT-3′ ACP 93 P53N3B-5′-GTCTACCAGGCATTCGCTTCATIIIIICTCCCC C CGT-3′ ACP 94 P53N4A-5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC G CG-3′ ACP 95 P53N4B-5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC C CG-3′ ACP 96 P53N5A-5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC G -3′ ACP 97 P53N5B-5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC C -3′ ACP 98 703N5′-ATTCTGATGGTGTGGATTGTG-3′ 99 SM703C5′-TCACAGAAGTATGCCAAGCGAIIIIIACCCTGGAGTAG ACGAAGA-3′ 100 SM703-A5′-GTCTACCAGGCATTCGCTTCATIIIIIGGTAC A GGGC-3′ 101 SM703-B5′-GTCTACCAGGCATTCGCTTCATIIIIIGGTAC C GGGC-3′ 102 028N5′-CCTTCTGTGCTTGATGCTTTT-3′ 103 SMO28C5′- TCACAGAAGTATGCCAAGCGAIIIIICAGGAAGGATGA GCATTTAG-3′ 104 SMO28-A5′-GTCTACCAGGCATTCGCTTCATIIIIITCCAA A CCAA-3′ 105 SMO28-B5′-GTCTACCAGGCATTCGCTTCATIIIIITCCAA C CCAA-3′ 106 695N5′-AGAAAAACCAGAGGCAGCTT-3′ 107 SM695C5′-TCACAGAAGTATGCCAAGCGAIIIIIAGCACAAACCAA AGACACAGT-3′ 108 SM695-A5′-GTCTACCAGGCATTCGCTTCATIIIII CCATT T TAGG-3′ 109 SM695-B5′-GTCTACCAGGCATTCGCTTCATIIIII CCATT G TAGG-3′ 110 679N5′-CTAGCTGCAAGTGACATCTCT-3′ 111 SM679C5′-TCACAGAGTATCCAAGCGIIIIITCAGTAAGAAG CCAGGAGAG-3′ 112 SM679-A5′-GTCTACCAGGCATTCGCTTCATIIIIICCAGA A CTTT-3′ 113 SM679-B5′-GTCTACCAGGCATTCGCTTCATIIIIICCAGA C CTTT-3′ 114 832N5′-TTTTGGGTGGAGGCTAACAT-3′ 115 SM832C5′-TCACAGAAGTATGCCAGCGAIIIIIAACGATGCAG ACACCACCA-3′ 116 SM832-A5′-GTCTACCAGGCATTCGCTTCATIIIIIGACTG G TAAA-3′ 117 SM832-B5′-GTCTACCAGGCATTCGCTTCATIIIIIGACTG A TAAA-3′ 118 880N5′-CTTCCACCAATACTCTTTTCC-3′ 119 SM880C5′-TCACAGAAGTATGCCAGCGAIIIIIGCATACACACA AGAGGCAGA-3′ 120 SM880-A5′-GTCTACCAGGCATTCGCTTCATIIIIIAAAGC C ATAA-3′ 121 SM880-B5′-GTCTACCAGGCATTCGCTTCATIIIIIAAAGC T ATAA-3′ S = G or C W = A or T V =A, G, or C N = A, G, C, or T I is deoxyinosine r is ribose d isdeoxyribose

TABLE 2 Differentially Expressed cDNA Fragments Cloned by the ACP of thePresent Invention Nomenclature Identity Homology DEG 1 Tropomyosin 2(beta) Mouse 92% DEG 2 Novel Novel DEG 3 Hypothetical protein (Tes gene)Mouse 99% DEG 4 Protease-6 Mouse 92% DEG 5 Novel Novel DEG 6 Cytochromec oxidase, subunit Vb Mouse 99% DEG 7 Hydroxylacyl-Coenzyme A Mouse 98%dehydrogense (Hadh) DEG 8 Troponin T2, cardiac (Tnnt2) Mouse 94% DEG 9RNA binding motif protein, Mouse 96% X chromosome DEG 10 Peroxiredoxin 6(Prdx6) Mouse 89% DEG 11 11 days or 13 days embryo cDNA Mouse 98%

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What is claimed is:
 1. An annealing control primer for improvingannealing specificity in nucleic acid amplification, which has thefollowing general formula:5′-X_(p)—Y_(q)—Z_(r)-3′ wherein X_(p) represents a 5′-end portionconsisting of a pre-selected arbitrary nucleotide sequence substantiallynot complementary to any site on a template nucleic acid; Y_(q)represents a regulator portion comprising at least three contiguousuniversal bases; Z_(r) represents a 3′-end portion having a hybridizingnucleotide sequence substantially complementary to a site on thetemplate nucleic acid to hybridize therewith; wherein p, q and rrepresent the number of nucleotides; and wherein X, Y and Z isdeoxyribonucleotide or ribonucleotide; wherein the regulator portion hasthe lowest T_(m) in the three portions of the primer, whereby theregulator portion is capable of regulating an annealing portion of theprimer in association with annealing temperatures.
 2. The annealingcontrol primer according to claim 1, wherein said universal base isselected from the group consisting of deoxyinosine, inosine,7-deaza-2′-deoxyinosine, 2-aza-2′-deoxyinosine, 2′-OMe inosine, 2′-Finosine, deoxy 3-nitropyrrole, 3-nitropyrrole, 2′-OMe 3-nitropyrrole,2′-F 3-nitropyrrole, 1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole,deoxy 5-nitroindole, 5-nitroindole, 2′-OMe 5-nitroindole, 2′-F5-nitroindole, deoxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxy4-aminobenzimidazole, 4-aminobenzimidazole, deoxy nebularine, 2′-Fnebularine, 2′-F 4-nitrobenzimidazole, PNA-5-introindole,PNA-nebularine, PNA-inosine, PNA-4-nitrobenzimidazole,PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino-nebularine,morpholino-inosine, morpholino-4-nitrobenzimidazole,morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole,phosphoramidate-nebularine, phosphoramidate-inosine,phosphoramidate-4-nitrobenzimidazole, phosphoramidate-3-nitropyrrole,2′-0-methoxyethyl inosine, 2′0-methoxyethyl nebularine,2′-0-methoxyethyl 5-nitroindole, 2′-0-methoxyethyl4-nitro-benzimidazole, 2′-0-methoxyethyl 3-nitropyrrole, andcombinations thereof.
 3. The annealing control primer according to claim2, wherein said universal base is deoxyinosine,1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole or 5-nitroindole. 4.The annealing control primer according to claim 1, wherein p representsan integer of 15 to
 60. 5. The annealing control primer according toclaim 1, wherein q represents an integer of 3 to
 15. 6. The annealingcontrol primer according to claim 1, wherein r represents an integer of6 to
 50. 7. The annealing control primer according to claim 1, wherein pis an integer of 15 to 60, q is an integer of 3 to 15 and r is aninteger of 6 to
 30. 8. The annealing control primer according to claim1, wherein X_(p) comprises a universal primer sequence.
 9. The annealingcontrol primer according to claim 1, wherein X_(p) comprises a sequenceor sequences recognized by a restriction endonuclease or restrictionendonucleases.
 10. The annealing control primer according to claim 1,wherein X_(p) comprises at least one nucleotide with a label fordetection or isolation.
 11. The annealing control primer according toclaim 1, wherein Z_(r) is a nucleotide sequence which hybridizes to thepolyadenosine (polyA) tail of an mRNA.
 12. The annealing control primeraccording to claim 1, wherein Z_(r) is a random nucleotide sequence. 13.The annealing control primer according to claim 1, wherein Z_(r) is anucleotide sequence substantially complementary to a consensus sequencefound in a gene family.
 14. The annealing control primer according toclaim 1, wherein Z_(r) is a degenerate nucleotide sequence selected froma plurality of combinations of nucleotides encoding a predeterminedamino acid sequence.
 15. The annealing control primer according to claim1, wherein Z_(r) comprises at least one nucleotode complementary toallelic site.
 16. The annealing control primer according to claim 1,wherein Z_(r) comprises at least one mismatch nucleotide to a targetnucleic acid for mutagenesis.